Recombinant adenoviruses and use thereof

ABSTRACT

The present invention relates to recombinant adenoviruses and vectors thereof. In particular, the adenoviruses are novel simian adenoviruses having a low seroprevalence and high immunogenicity relative to other adenoviruses and vectors thereof. The invention also provides methods for production of the adenoviruses and for the treatment of diseases by administering the adenoviral vector(s) to a subject (e.g., a human).

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant Nos.AI078526, AI096040, and OD011170, awarded by the National Institutes ofHealth (NIH). The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 24, 2019, isnamed 01948-219003_Sequence_Listing_12_24_19_ST25 and is 600,226 bytesin size.

BACKGROUND OF THE INVENTION

Recombinant adenoviral vectors have been used in the development ofvaccines. To date, approximately 55 different adenovirus serotypes havebeen identified. The subgroup C adenoviruses have been most extensivelystudied for applications such as vaccination and gene therapy.Adenovirus serotypes 2 and 5 (Ad2 and Ad5), in particular, are widelyused in the field. Importantly, Ad5 vector-based vaccines have beenshown to elicit potent and protective immune responses in a variety ofanimal models. Moreover, large-scale clinical trials for HIV vaccinationusing Ad5-based recombinant vectors are ongoing (see, e.g., WO 01/02607;WO 02/22080; Shiver et al., Nature. 415:331-335, 2002; Letvin et al.,Annu. Rev. Immunol. 20:73-99, 2002; and Shiver and Emini, Annu. Rev.Med. 55:355, 2004).

The usefulness of recombinant Ad5 vector-based vaccines for HIV andother pathogens, however, may be limited due to high pre-existinganti-Ad5 immunity in human populations. The presence of anti-Ad5immunity has been correlated with a reduction in the immunogenicity ofAd5-based vaccines in studies in mice and rhesus monkeys. Early datafrom phase-1 clinical trials show that this problem may also occur inhumans. Although both Ad5-specific neutralizing antibodies (NAbs) andCD8⁺ T lymphocytes contribute to anti-Ad5 immunity, the Ad5-specificNAbs appear to play the primary role in this process (Sumida et al., J.Virol., 174:7179-7185, 2004).

Accordingly, there is an unmet need in the field for alternativeadenoviral vectors that have low seroprevalence and potentimmunogenicity.

SUMMARY OF THE INVENTION

The entire genomes of three novel simian adenoviruses (sAds), sAd4287,sAd4310A, and sAd4312, have been identified and their entire genomesdetermined. These adenoviruses exhibit both surprisingly lowseroprevalence and potent immunogenicity, which suggests that theseviruses may be useful as novel vaccine vector candidates. In a firstaspect, this invention features isolated polynucleotides including anucleotide sequence that is at least 90% identical (e.g., at least 91%,92%, 93%, or 94% identical), at least 95% identical (e.g., at least 96%,97%, 98%, or 99% identical), or 100% identical to all or a portion ofany one of SEQ ID NOs: 1-3, or its complement. SEQ ID NOs: 1, 2, and 3are the full-length genome sequence of wild-type sAd4287, sAd4310A, andsAd4312, respectively. The isolated polynucleotides of the invention mayinclude at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, or 35000 ormore contiguous or non-contiguous nucleotides of a referencepolynucleotide molecule (e.g., SEQ ID NOs: 1-3).

In some embodiments, the isolated polynucleotides of the inventioninclude a nucleotide sequence that is at least 90% identical (e.g., atleast 91%, 92%, 93%, or 94% identical), at least 95% identical (e.g., atleast 96%, 97%, 98%, or 99% identical), or 100% identical to all or aportion of any one of SEQ ID NOs: 4-12, or its complement. SEQ NOs: 4-12feature the nucleotide sequences encoding the fiber-1, fiber-2, andhexon proteins of wild-type sAd4287, sAd4310A, and sAd4312. Accordingly,in some embodiments, the nucleotide sequence encoding all or a portionof the fiber-1 protein can be at least 90% identical (e.g., at least91%, 92%, 93%, or 94% identical), at least 95% identical (e.g., at least96%, 97%, 98%, or 99% identical), or 100% identical to the nucleotidesequence encoding the fiber-1 protein of wild-type sAd4287, sAd4310A, orsAd4312, which corresponds to SEQ ID NO: 4, 5, and 6, respectively. Insome embodiments, the nucleotide sequence encoding all or a portion ofthe fiber-2 protein can be at least 90% identical (e.g., at least 91%,92%, 93%, or 94% identical), at least 95% identical (e.g., at least 96%,97%, 98%, or 99% identical), or 100% identical to the nucleotidesequence encoding the fiber-2 protein of wild-type sAd4287, sAd4310A, orsAd4312, which corresponds to SEQ ID NO: 7, 8, and 9, respectively. Insome embodiments, the nucleotide sequence encoding all or a portion ofthe hexon protein can be at least 90% identical (e.g., at least 91%,92%, 93%, or 94% identical), at least 95% identical (e.g., at least 96%,97%, 98%, or 99% identical), or 100% identical to the nucleotidesequence encoding the hexon protein of wild-type sAd4287, sAd4,310A, orsAd4312, which corresponds to SEQ ID NO: 10, 11, and 12, respectively.In some embodiments, the nucleotide sequence can be at least 90%identical (e.g., at least 91%, 92%, 93%, or 94% identical), at least 95%identical (e.g., at least 96%, 97%, 98%, or 99% identical), or 100%identical to all or a portion of one or more hexon protein hypervariableregions (HVRs) (e.g., HVR1 (nt 403 to nt 489), HVR2 (nt 520 to nt 537),HVR3 (nt 592 to nt 618), HVR4 (nt 706 to nt 744), HVR5 (nt 763 to 786),HVR6 (nt 856 to nt 874), and/or HVR7 (nt 1201 to nt 1296) of sAd4287hexon protein (SEQ ID NO: 10); HVR1 (nt 403 to nt 477), HVR2 (nt 505 tont 516), HVR3 (nt 571 to nt 591), HVR4 (nt 679 to nt 690), HVR5 (nt 709to 735), HVR6 (nt 805 to nt 816), and/or HVR7 (nt 1144 to nt 1236) ofsAd4310A hexon protein (SEQ ID NO: 11); or HVR1 (nt 403 to nt 474), HVR2(nt 505 to nt 522), HVR3 (nt 577 to nt 597), HVR4 (nt 685 to nt 726),HVR5 (nt 748 to 777), HVR6 (nt 847 to nt 864), and/or HVR7 (nt 1192 tont 1284) of sAd4312 hexon protein (SEQ ID NO: 12)).

In some embodiments, the one or more nucleotide sequences encoding oneor more hexon protein hypervariable regions (HVRs) of the invention havebeen substituted with that of another virus (e.g., HVR1 (nt 403 to nt489), HVR2 (nt 520 to nt 537), HVR3 (nt 592 to nt 618), HVR4 (nt 706 tont 744). HVR5 (nt 763 to 786), HVR6 (nt 856 to nt 874), and/or HVR7 (nt1201 to nt 1296) of sAd4287 hexon protein (SEQ ID NO: 10); HVR1 (nt 403to nt 477). HVR2 (nt 505 to nt 516), HVR3 (nt 571 to nt 591), HVR4 (nt679 to nt 690), HVR5 (nt 709 to 735), HVR6 (nt 805 to nt 816), and/orHVR7 (nt 1144 to nt 1236) of sAd4310A hexon protein (SEQ ID NO: 11); orHVR1 (nt 403 to nt 474), HVR2 (nt 505 to nt 522), HVR3 (nt 577 to nt597), HVR4 (nt 685 to nt 726), HVR5 (nt 748 to 777), HVR6 (nt 847 to nt864), and/or HVR7 (nt 1192 to nt 1284) of sAd4312 hexon protein (SEQ IDNO: 12)) substituted with the corresponding HVR sequences of one or moreother viruses, e.g., an adenovirus, e.g., an adenovirus that has a lowerseroprevalence compared to that of Ad5, such as subgroup B (Ad11, Ad34,Ad35, and Ad50) and subgroup D (Ad15, Ad24, Ad26, Ad48, and Ad49)adenoviruses as well as simian adenoviruses (e.g., Pan9, also known asAdC68)). In other embodiments, the nucleotide sequence includes anadenoviral vector backbone of Ad5, Ad11, Ad15, Ad24, Ad26, Ad34, Ad48,Ad49, Ad50, or Pan9/AdC68 having a substitution of all or a portion ofone or more of the above hexon HVRs of sAd4287, sAd4310A, and/orsAd4312.

In some embodiments, the isolated polynucleotides of the inventioninclude a nucleotide sequence that is at least 90% identical (e.g., atleast 91%, 92%, 93%, or 94% identical), at least 95% identical (e.g., atleast 96%, 97%, 98%, or 99% identical), or 100% identical to all or aportion of any one of SEQ ID NOs: 13-18, or its complement. SEQ ID NOs:13-18 feature the nucleotide sequences encoding the knob domain of thefiber-1 and fiber-2 proteins of wild-type sAd4287, sAd4310A, andsAd4312. In some embodiments, the nucleotide sequence encoding all or aportion of the knob domain of fiber-1 can be at least 90% identical(e.g., at least 91%, 92%, 93%, or 94% identical), at least 95% identical(e.g., at least 96%, 97%, 98%, or 99% identical), or 100% identical tothe nucleotide sequence encoding the knob domain of the fiber-1 proteinof wild-type sAd4287, sAd4310A, or sAd4312, which corresponds to SEQ IDNO: 13, 14, and 15, respectively. In some embodiments, the nucleotidesequence encoding all or a portion of the knob domain of fiber-2 can beat least 90% identical (e.g., at least 91%, 92%, 93%, or 94% identical),at least 95% identical (e.g., at least 96%, 97%, 98%, or 99% identical),or 100% identical to the nucleotide sequence encoding the knob domain ofthe fiber-2 protein of wild-type sAd4287, sAd4310A, or sAd4312, whichcorresponds to SEQ ID NO: 16, 17, and 18, respectively. In someembodiments, one or more nucleotide sequences encoding a knob domain ofa fiber protein (e.g., a fiber-1 or fiber-2 protein) of the invention(SEQ ID NOs: 13-18) have been substituted with that of another virus.

In a second aspect, the invention features recombinant vectors includingan isolated polynucleotide of the invention, the recombinant vectorsincluding a nucleotide sequence that is at least 90% identical (e.g., atleast 91%, 92%, 93%, or 94% identical), at least 95% identical (e.g., atleast 96%. 97%, 98%, or 99% identical), or 100% identical to all or aportion of any one of SEQ ID NOs: 34-51. In some embodiments, the vectoris an sAd4297 adenoviral vector including all or a portion of any one ofSEQ ID NOs: 34-39. In some embodiments, the vector is an sAd4310Aadenoviral vector including all or a portion of any one of SEQ ID NOs:40-45. In some embodiments, the vector is an sAd4312 adenoviral vectorincluding all or a portion of any one of SEQ ID NOs: 46-51. In otherembodiments, more than one (e.g., 2, 3, or 4) of the vectors describedby SEQ ID NOs: 34-51 may be used to establish a plasmid system for thegeneration of a recombinant adenovirus of the invention.

In an embodiment of the first or second aspect of the invention, theisolated polynucleotides and/or recombinant vectors are used to generaterecombinant adenoviruses wherein all or a portion of the adenoviruses isderived from any one of SEQ ID NOs: 1-3. In some embodiments, therecombinant adenovirus includes an isolated polynucleotide including adeletion in or of the E1 region (e.g., nt 474 to nt 3085 of sAd4287 (SEQID NO: 1); nt 474 to nt 3088 of sAd4310A (SEQ ID NO: 2); and nt 487 tont 3100 of sAd4312 (SEQ ID NO: 3)). A recombinant adenoviral vector thatincludes this deletion is rendered replication-defective. In someembodiments, the replication-defective virus may also include a deletionin or of the E3 region (e.g., nt 25973 to nt 28596 of sAd4287 (SEQ IDNO: 1); nt 25915 to nt 28496 of sAd4310A (SEQ ID NO: 2); and nt 25947 tont 28561 of sAd4312 (SEQ ID NO: 3)) and/or E4 region (e.g., nt 31852 tont 34752 of sAd4287 (SEQ ID NO: 1); nt 31750 to nt 34048 of sAd4310A(SEQ ID NO: 2); and nt 31818 to nt 34116 of sAd4312 (SEQ ID NO: 3)). Inother embodiments, the recombinant adenovirus includes one or more ofthe E1, E3, and/or E4 regions and is replication-competent.

According to a preferred embodiment, the recombinant adenovirus furtherincludes a heterologous nucleotide sequence encoding an antigenic ortherapeutic gene product of interest, or fragment thereof. In someembodiments, the antigenic gene product, or fragment thereof, includes abacterial, viral, parasitic, or fungal protein, or fragment thereof.

The bacterial protein, or fragment thereof, may be from Mycobacteriumtuberculosis, Mycobacterium Bovis, Mycobacterium africanum,Mycobacterium microti, Mycobacterium leprae, Pseudomonas aeruginosa,Salmonella typhimurium, Escherichia coil, Klebsiella pneumoniae,Streptococcus pneumoniae, Staphylococcus aureus, Francisella tularensis,Brucella, Burkholderia mallei, Yersinia pestis, Corynebacteriumdiphtheria, Neisseria meningitidis, Bordetella pertussis, Clostridiumtetani, or Bacillus anthracis. Examples of preferred gene products, orfragments thereof, from Mycobacterium strains include 10.4, 85A, 85E,85C, CFP-10, Rv3871, and ESAT-6 gene products or fragments thereof.

The viral protein, or fragment thereof, may be from a virus of theRetroviridae family, which includes the human immunodeficiency virus(HIV; e.g., types 1 and 2), and human T-lymphotropic virus Types I andII (HTLV-1 and HTLV-2, respectively); Flaviviridae family (e.g., amember of the Flavivirus, Pestivirus, and Hepacivirus genera), whichincludes the hepatitis C virus (HCV), Yellow fever virus, tick-borneviruses, such as the Gadgets Gully virus, Kadam virus, Kyasanur Forestdisease virus, Langat virus, Omsk hemorrhagic fever virus, Powassanvirus, Royal Farm virus, Karshi virus, tick-borne encephalitis virus,Neudoerfl virus, Sofjin virus, Louping ill virus and the Negishi virus;seabird tick-borne viruses, such as the Meaban virus, Saumarez Reefvirus, and the Tyuleniy virus; mosquito-borne viruses, such as the Aroavirus, dengue virus, Kedougou virus, Cacipacore virus, Koutango virus,Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louisencephalitis virus, Usutu virus, West Nile virus, Yaounde virus,Kokobera virus, Bagaza virus, Ilheus virus, Israel turkeymeningoencephalo-myelitis virus, Ntaya virus, Tembusu virus, Zika virus,Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus,Sepik virus, Uganda S virus, Wesselsbron virus, yellow fever virus; andviruses with no known arthropod vector, such as the Entebbe bat virus,Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modocvirus, Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Careyisland virus, Dakar bat virus, Montana myotis leukoencephalitis virus,Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, and the Cellfusing agent virus; Arenaviridae family, which includes the Ippy virus,Lassa virus (e.g., the Josiah, LP, or GA391 strain), lymphocyticchoriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amaparivirus, Flexal virus, Guanarito virus, Junin virus, Latino virus, Machupovirus, Cliveros virus, Paraná virus, Pichinde virus, Pirital virus,Sabiá virus, Tacaribe virus, Tamiami virus, Whitewater Arroyo virus,Chapare virus, and Lujo virus; Bunyaviridae family (e.g., a member ofthe Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirus genera),which includes the Hantaan virus, Sin Nombre virus, Dugbe virus,Bunyamwera virus, Rift Valley fever virus, La Crosse virus, Punta Torovirus (PTV), California encephalitis virus, and Crimean-Congohemorrhagic fever (CCHF) virus; Filoviridae family, which includes theEbola virus (e.g., the Zaire, Sudan, Ivory Coast, Reston, and Ugandastrains) and the Marburg virus (e.g., the Angola, Ci67, Musoke, Popp,Ravn and Lake Victoria strains); Togaviridae family (e.g., a member ofthe Alphavirus genus), which includes the Venezuelan equine encephalitisvirus (VEE), Eastern equine encephalitis virus (EEE), Western equineencephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forestvirus, Ross River virus, Barman Forest virus, O'nyong'nyong virus, andthe chikungunya virus; Poxviridae family (e.g., a member of theOrthopoxvirus genus), which includes the smallpox virus, monkeypoxvirus, and vaccinia virus; Herpesviridae family; which includes theherpes simplex virus (HSV; types 1, 2, and 6), human herpes virus (e.g.,types 7 and 8), cytomegalovirus (CMV), Epstein-Barr virus (EBV),Varicella-Zoster virus, and Kaposi's sarcoma associated-herpesvirus(KSHV); Orthomyxoviridae family, which includes the influenza virus (A,B, and C), such as the H5N1 avian influenza virus or H1N1 swine flu;Coronaviridae family, which includes the severe acute respiratorysyndrome (SARS) virus; Rhabdoviridae family, which includes the rabiesvirus and vesicular stomatitis virus (VSV); Paramyxoviridae family,which includes the human respiratory syncytial virus (RSV), Newcastledisease virus, hendravirus, nipahvirus, measles virus, rinderpest virus,canine distemper virus, Sendai virus, human parainfluenza virus (e.g.,1, 2, 3, and 4), rhinovirus, and mumps virus; Picornaviridae family,which includes the poliovirus, human enterovirus (A, B, C, and D),hepatitis A virus, and the coxsackievirus; Hepadnaviridae family, whichincludes the hepatitis B virus; Papillomaviridae family, which includesthe human papiliomavirus; Parvoviridae family, which includes theadeno-associated virus; Astroviridae family, which includes theastrovirus; Polyomaviridae family, which includes the JC virus, BKvirus, and SV40 virus; Calciviridae family, which includes the Norwalkvirus; or Reoviridae family, which includes the rotavirus. In apreferred embodiment, the viral protein, or fragment thereof, is fromhuman immunodeficiency virus (HIV), human papiliomavirus (HPV),hepatitis A virus (Hep A), hepatitis B virus (HBV), hepatitis C virus(HCV), Variola major, Variola minor, monkeypox virus, measles virus,rubella virus, mumps virus, varicella zoster virus (VZV), poliovirus,rabies virus, Japanese encephalitis virus, herpes simplex virus (HSV),cytomegalovirus (CMV), rotavirus, influenza, Ebola virus, yellow fevervirus, or Marburg virus. In a most preferred embodiment, the viralprotein, or fragment thereof, from HIV is Gag, Pol, Env, Nef, Tat, Rev,Vif, Vpr, or Vpu.

The parasitic protein, or fragment thereof, may be from Toxoplasmagondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale,Plasmodium malariae, Trypanosome spp., or Legionella spp. Examples ofparticularly preferred parasitic proteins that may be cloned into thevectors of the present invention include those from Plasmodiumfalciparum, such as the circumsparozoite (CS) protein and Liver SpecificAntigens 1 or 3 (SA-1 or LSA-3).

The fungal protein, or fragment thereof, may be from Aspergillus,Blastomyces dermatitidis, Candida, Coccidioides immitis, Cryptococcusneoformans, Histoplasma capsulatum var. capsulatum, Paracoccidioidesbrasiliensis, Sporothrix schenckii, Zygomycetes spp., Absidiacorymbifera, Rhizomucor pusillus, or Rhizopus arrhizus. Examples offungal gene products, or fragments thereof, include any cell wallmannoprotein (e.g., Afmp1 of Aspergillus fumigatus) or surface-expressedglycoprotein (e.g., SOWgp of Coccidioides immitis).

The therapeutic gene products, or fragments thereof, may be interferon(IFN) proteins, Factor VIII, Factor IX, erythropoietin, alpha-1antitrypsin, calcitonin, glucocerebrosidase, growth hormone, low densitylipoprotein (LDL), receptor IL-2 receptor and its antagonists, insulin,globin, immunoglobulins, catalytic antibodies, the interleukins,insulin-like growth factors, superoxide dismutase, immune respondermodifiers, parathyroid hormone and interferon, nerve growth factors,tissue plasminogen activators, and/or colony stimulating factors, orfragments thereof.

A third aspect of the invention features a method of treating a subject(e.g., a human) having a disease (e.g., HIV or cancer) by administeringa recombinant sAd adenovirus vector of the second aspect of theinvention to the subject. In a preferred embodiment, the recombinant sAdadenovirus of the invention includes an antigenic gene product, orfragment thereof, that promotes an immune response against an infectiveagent in a subject at risk of exposure to, or exposed to, the infectiveagent. In some embodiments, the infective agent is a bacterium, a virus,a parasite, or a fungus, such as those described above. In onenon-limiting example, the administration of a sAd adenovirus of theinvention expressing an HIV Gag protein, or fragment thereof, to anHIV-positive subject or a subject with acquired immune deficiencysyndrome (AIDS) can stimulate an immune response in the subject againstHIV, thereby treating the subject. In another embodiment, therecombinant sAd adenovirus of the invention includes a therapeutic geneproduct, or fragment thereof, such as an interferon (IFN) protein, orfragment thereof, that provides therapy to a subject having a diseasecaused by a non-infective agent, such as cancer, by stimulating afavorable immune response in the subject against neoplasia and/or byproviding gene therapy, thereby treating the subject. Other non-limitingexamples of diseases that may be treated include any human healthdisease, such as tuberculosis, leprosy, typhoid fever, pneumonia,meningitis, staphylococcal scalded skin syndrome (SSSS), Ritter's,disease, tularemia (rabbit fever), brucellosis, Glanders disease,bubonic plague, septicemic plague, pneumonic plague, diphtheria,pertussis (whooping cough), tetanus, anthrax, hepatitis, smallpox,monkeypox, measles, mumps, rubella, chicken pox, polio, rabies, Japaneseencephalitis, herpes, mononucleosis, influenza, Ebola virus disease,hemorrhagic fever, yellow fever, Marburg virus disease, toxoplasmosis,malaria, trypanosomiasis, legionellosis, aspergillosis, blastomycosis,candidiasis (thrush), coccidioidomycosis, cryptococcosis,histoplasmosis, paracoccidioidomycosis, sporotrichosis, or sinus-orbitalzygomycosis. Treatment of these diseases may be by administration of arecombinant sAd vector of the invention that encodes or expresses on itssurface an immune response-stimulating antigen from the selectedinfective agent.

In some embodiments, the recombinant adenovirus or adenoviral vector isadministered Intramuscularly, intravenously, intradermally,percutaneously, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostatically, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, peritoneally, subcutaneously,subconjunctivally, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularly, orally, topically, locally, byinhalation, by injection, by infusion, by continuous infusion, bylocalized perfusion bathing target cells directly, by catheter, bylavage, by gavage, in cremes, or in lipid compositions. In one preferredembodiment, the recombinant adenovirus or adenoviral vector isadministered as a pharmaceutical composition that includes apharmaceutically acceptable carrier, diluent, or excipients, and mayoptionally include an adjuvant. In some embodiments, the subject isadministered at least one dose (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore doses) of the composition. In other embodiments, the subject isadministered at least two doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore doses) of the composition. In yet another embodiment, thepharmaceutical composition is administered to the subject as a primeboost or in a prime boost regimen. The subject can be administered atleast about 1×10³ viral particles (vp)/dose or between 1×10¹ and 1×10¹⁴vp/dose, preferably between 1×10³ and 1×10¹² vp/dose, and morepreferably between 1×10⁵ and 1×10¹⁴ vp/dose. The pharmaceuticalcomposition may be administered, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 30, 35, 40, 45, 50, 55, or 60 minutes, 2, 4, 6, 10, 15, or24 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, or even 3, 4, or 6months pre-exposure or pre-diagnosis, or may be administered to thesubject 15-30 minutes or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 20, 24, 48,or 72 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, 3, 4, 6, or 9months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 years or longerpost-diagnosis or post-exposure or to the infective agent. When treatingdisease (e.g., AIDS or cancer), the pharmaceutical compositions of theinvention may be administered to the subject either before theoccurrence of symptoms or a definitive diagnosis or after diagnosis orsymptoms become evident. The pharmaceutical composition may beadministered, for example, immediately after diagnosis or the clinicalrecognition of symptoms or 2, 4, 6, 10, 15, or 24 hours, 2, 3, 5, or 7days, 2, 4, 6 or 8 weeks, or even 3, 4, or 6 months after diagnosis ordetection of symptoms.

In a fourth aspect, the invention features a method of producing arecombinant adenovirus of the invention that includes culturing a cellin a suitable medium; transfecting the cell with an isolatedpolynucleotide of the first aspect of the invention or a recombinantvector of the second aspect of the invention; allowing replication ofthe polynucleotide or vector in the cell; and harvesting the producedrecombinant adenovirus from the medium and/or cell. In some embodiments,the cell is a bacterial, plant, or mammalian cell. In a preferredembodiment, the mammalian cell is a PER.55K cell or a Chinese hamsterovary (CHO) cell.

Definitions

By “adenovirus” is meant a medium-sized (90-100 nm), nonenvelopedicoshedral virus that includes a capsid and a double-stranded linear DNAgenome. The adenovirus can be a naturally occurring, but isolated,adenovirus (e.g., sAd4287, sAd4310A, or sAd4312) or a recombinantadenovirus (e.g., replication-defective or replication competentsAd4287, sAd4310A, or sAd4312, or a chimeric variant thereof).

As used herein, by “administering” is meant a method of giving a dosageof a pharmaceutical composition (e.g., a recombinant adenovirus of theinvention) to a subject. The compositions utilized in the methodsdescribed herein can be administered, for example, intramuscularly,intravenously, intradermally, percutaneously, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrapleurally, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, topically,intratumorally, peritoneally, subcutaneously, subconjunctivally,intravesicularlly, mucosally, intrapericardially, intraocularly, orally,topically, locally, by inhalation, by injection, by infusion, bycontinuous infusion, by localized perfusion bathing target cellsdirectly, by catheter, by lavage, by gavage, in cremes, or in lipidcompositions. The preferred method of administration can vary dependingon various factors (e.g., the components of the composition beingadministered and the severity of the condition being treated).

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

By “deletion” of an adenoviral genomic region is meant the partial orcomplete removal, the disruption (e.g., by an insertion mutation), orthe functional inactivation (e.g., by a missense mutation) of aspecified genomic region (e.g., the E1, E2, E3, and/or E4 region), orany specific open-reading frame within the specified region.

By “gene product” is meant to include mRNAs or other nucleic acids(e.g., microRNAs) transcribed from a gene as well as polypeptidestranslated from those mRNAs. In some embodiments, the gene product isfrom a virus (e.g., HIV) and many include, for example, any one or moreof the viral proteins, or fragments thereof, described in, for example,pending U.S. Pub. No, 2012/0076812. In some embodiments, the geneproduct is a therapeutic gene product, including, but not limited to,interferon proteins, Factor VIII, Factor IX, erythropoietin, alpha-1antitrypsin, calcitonin, glucocerebrosidase, growth hormone, low densitylipoprotein (LDL), receptor IL-2 receptor and its antagonists, insulin,globin, immunoglobulins, catalytic antibodies, the interleukins,insulin-like growth factors, superoxide dismutase, immune respondermodifiers, parathyroid hormone and interferon, nerve growth factors,tissue plasminogen activators, and colony stimulating factors.

By “heterologous nucleic acid molecule” is meant any exogenous nucleicacid molecule that can be incorporated into, for example, an adenovirusof the invention, or polynucleotide or vector thereof, for subsequentexpression of a gene product of interest, or fragment thereof, encodedby the heterologous nucleic acid molecule. In a preferred embodiment,the heterologous nucleic acid molecule encodes an antigenic ortherapeutic gene product, or fragment thereof, that is a bacterial,viral, parasitic, or fungal protein, or fragment thereof (e.g., anucleic acid molecule encoding one or more HIV or SIV Gag, Pol, Env,Net, Tat, Rev, Vif, Vpr, or Vpu gene products, or fragments thereof).The heterologous nucleic acid molecule is one that is not normallyassociated with the other nucleic acid molecules found in the wild-typeadenovirus.

By “isolated” is meant separated, recovered, or purified from acomponent of its natural environment.

By “pharmaceutical composition” is meant any composition that contains atherapeutically or biologically active agent, such as a recombinantadenoviral vector of the invention, preferably including a heterologousnucleotide sequence encoding an antigenic or therapeutic gene product ofinterest, or fragment thereof, that is suitable for administration to asubject and that treats a disease (e.g., cancer or AIDS) or reduces orameliorates one or more symptoms of the disease. For the purposes ofthis invention, pharmaceutical compositions include vaccines, andpharmaceutical compositions suitable for delivering a therapeutic orbiologically active agent can include, for example, tablets, gelcaps,capsules, pills, powders, granulates, suspensions, emulsions, solutions,gels, hydrogels, oral gels, pastes, eye drops, ointments, creams,plasters, drenches, delivery devices, suppositories, enemas,injectables, implants, sprays, or aerosols. Any of these formulationscan be prepared by well-known and accepted methods of art. See, forexample, Remington: The Science and Practice of Pharmacy (21^(st) ed.),ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopediaof Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare,2006, each of which is hereby incorporated by reference.

By “pharmaceutically acceptable diluent, excipient, carrier, oradjuvant” is meant a diluent, excipient, carrier, or adjuvant which isphysiologically acceptable to the subject while retaining thetherapeutic properties of the pharmaceutical composition with which itis administered. One exemplary pharmaceutically acceptable carrier isphysiological saline. Other physiologically acceptable diluents,excipients, carriers, or adjuvants and their formulations are known toone skilled in the art (see, e.g., U.S. Pub. No. 2012/0076812).

By “portion” or “fragment” is meant a part of a whole. A portion maycomprise at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ofthe entire length of an polynucleotide or polypeptide sequence region.For polynucleotides, for example, a portion may include at least 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 15000, 20000, 25000, 30000, 35000 or more contiguousnucleotides of a reference polynucleotide molecule. For polypeptides,for example, a portion may include at least 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 125, 150, 175, 200,225, 250, 275, 300, or 350 or more contiguous amino acids of a referencepolypeptide molecule.

By “promotes an immune response” is meant eliciting a humoral response(e.g., the production of antibodies) or a cellular response (e.g., theactivation of T cells, macrophages, neutrophils, and natural killercells) directed against, for example, one or more infective agents(e.g., a bacterium, virus, parasite, fungus, or combination thereof) orprotein targets in a subject to which the pharmaceutical composition(e.g., a vaccine) has been administered.

By “recombinant” with respect to a vector or virus, is meant a vector orvirus that has been manipulated in vitro, such as a vector or virus thatincludes a heterologous nucleotide sequence (e.g., a sequence encodingan antigenic or therapeutic gene product) or a vector or virus bearingan alteration, disruption, or deletion in a viral E1, E3, and/or E4region relative to a wild-type viral E1, E3, and/or E4 region.

By “sequence identity” or “sequence similarity” is meant that theidentity or similarity between two or more amino acid sequences, or twoor more nucleotide sequences, is expressed in terms of the identity orsimilarity between the sequences. Sequence identity can be measured interms of “percentage (%) identity,” wherein the higher the percentage,the more identity shared between the sequences. Sequence similarity canbe measured in terms of percentage similarity (which takes into accountconservative amino acid substitutions); the higher the percentage, themore similarity shared between the sequences. Homologs or orthologs ofnucleic acid or amino acid sequences possess a relatively high degree ofsequence identity/similarity when aligned using standard methods.Sequence identity may be measured using sequence analysis software onthe default setting (e.g., Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705). Such software may matchsimilar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications.

A “subject” is a vertebrate, such as a mammal (e.g., primates andhumans). Mammals also include, but are not limited to, farm animals(such as cows), sport animals (e.g., horses), pets (such as cats, anddogs), mice, and rats. A subject to be treated according to the methodsdescribed herein (e.g., a subject having a disease such as cancer and/ora disease caused by an infective agent, e.g., a bacterium, virus,fungus, or parasite) may be one who has been diagnosed by a medicalpractitioner as having such a condition. Diagnosis may be performed byany suitable means. A subject in whom the development of an infection isbeing prevented may or may not have received such a diagnosis. Oneskilled in the art will understand that a subject to be treatedaccording to the present invention may have been subjected to standardtests or may have been identified, without examination, as one at highrisk due to the presence of one or more risk factors (e.g., exposure toa biological agent, such as a virus).

As used herein, and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, such as clinicalresults. Beneficial or desired results can include, but are not limitedto, alleviation or amelioration of one or more symptoms or conditions;diminishment of extent of disease, disorder, or condition; stabilization(i.e., not worsening) of a state of disease, disorder, or condition;prevention of spread of disease, disorder, or condition; delay orslowing the progress of the disease, disorder, or condition;amelioration or palliation of the disease, disorder, or condition; andremission (whether partial or total), whether detectable orundetectable. “Palliating” a disease, disorder, or condition means thatthe extent and/or undesirable clinical manifestations of the disease,disorder, or condition are lessened and/or time course of theprogression is slowed or lengthened, as compared to the extent or timecourse in the absence of treatment.

The term “vaccine,” as used herein, is defined as material used toprovoke an immune response and may confer immunity after administrationof the vaccine to a subject.

By “vector” is meant a composition that includes one or more genes(non-structural or structural), or fragments thereof, from a viralspecies, such as an adenoviral species (e.g., sAd4287, sAd4310A, orsAd4312), that may be used to transmit one or more heterologous genesfrom a viral or non-viral source to a host or subject. The nucleic acidmaterial of the viral vector may be encapsulated, e.g., in a lipidmembrane or by structural proteins (e.g., capsid proteins), that mayinclude one or more viral polypeptides (e.g., a glycoprotein). The viralvector can be used to infect cells of a subject, which, in turn,promotes the translation of the heterologous gene(s) of the viral vectorinto a protein product.

The term “virus,” as used herein, is defined as an infectious agent thatis unable to grow or reproduce outside a host cell and that infectsmammals (e.g., humans) or birds.

Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic map of the genomic organization of sAd4287.

FIG. 2 is a schematic map of plasmid sAdApt4287.Empty.

FIG. 3 is a schematic map of plasmid pBr/sAd4287.pIX-pV.

FIG. 4 is a schematic map of plasmid pBr/sAd4287.PsiI-rITR.

FIG. 5 illustrates the cloning strategy used to obtain plasmidpBr/sAd4287.PsiI-rITR.dE3 and a schematic map ofpBr/sAd42.87.PsiI-rITR.dE3 relative to that of its parental plasmidpBr/sAd4287.PsiI-rITR.

FIG. 6 shows a schematic map of plasmid pBr/sAd4287.PsiI-rITR.dE3.dE4relative to that of its parental plasmid pBr/sAd4287.PsiI-rITR.dE3.

FIG. 7 illustrates the cloning strategy used to obtain plasmidsAdApt4287.E1btg.Empty and a schematic map of sAdApt4287.E1btg.Emptyrelative to that of its parental plasmid sAdApt4287.Empty.

FIG. 8 is a schematic map of the genomic organization of sAd4310 #13-1(sAd4310A).

FIG. 9 is a schematic map of plasmid sAdApt4310A.Empty.

FIG. 10 is a schematic map of plasmid pBr/sAd4310A.pIX-pV.

FIG. 11 is a schematic map of plasmid pBr/sAd4310A.RsrII-rITR.

FIG. 12 shows a schematic map of pBr/sAd4310A.RsrII-rITR.dE3 relative tothat of its parental plasmid pBr/sAd4310A.RsrII-rITR.

FIG. 13 shows a schematic map of plasmid pBr/sAd4310A.RsrII-rITR.dE3.dE4relative to that of its parental plasmid pBr/sAd4310A.RsrII-rITR.dE3.

FIG. 14 illustrates the cloning strategy used to obtain plasmidsAdApt4310A.E1btg.Empty and a schematic map of sAdApt4310A.E1btg.Emptyrelative to that of its parental plasmid sAdApt4310A.Empty.

FIG. 15 is a schematic map of the genomic organization of sAd4312.

FIG. 16 is a schematic map of plasmid sAdApt4312.Empty.

FIG. 17 is a schematic map of plasmid pBr/sAd4312.pIX-pV.

FIG. 18 is a schematic map of plasmid pBr/sAd4312.pV-rITR.

FIG. 19 illustrates the cloning strategy used to obtain plasmidpBr/sAd4312.pV-rITR.dE3 and a schematic map of pBr/sAd4312.pV-rITR.dE3relative to that of its parental plasmid pBr/sAd4312.pV-rITR.

FIG. 20 shows a schematic map of plasmid pBr/sAd4312.pV-rITR.dE3.dE4relative to that of its parental plasmid pBr/sAd4312.pV-rITR.dE3.

FIG. 21 is a schematic map of plasmid sAdApt4312.E1btg.Empty.

FIG. 22A is a pie chart showing the relative sAd4287-specificneutralizing antibody (NAb) responses in sub-Saharan humans (n=144; top)and rhesus monkeys (n=108; bottom). The relative number of individualsthat fall within each of the four NAb titer categories (<18=negative,18-200=low, 201-1000=high, and >1000=high), as assessed byluciferase-based virus neutralization assays, is shown.

FIG. 22B is a pie chart showing the relative sAd4310A-specificneutralizing antibody (NAb) responses in sub-Saharan humans (n=144; top)and rhesus monkeys (n=108; bottom). The relative number of individualsthat fall within each of the four NAb titer categories (<18=negative,18-200=low, 201-1000=high and >1000=high), as assessed byluciferase-based virus neutralization assays, is shown.

FIG. 22C is a pie chart showing the relative sAd4312-specificneutralizing antibody (NAb) responses in sub-Saharan humans (n=144; top)and rhesus monkeys (n=108; bottom). The relative number of individualsthat fall within each of the four NAb titer categories (<18=negative,18-200=low, 201-1000=high, and >1000=high), as assessed byluciferase-based virus neutralization assays, is shown.

FIG. 23A is a graph showing the cellular responses induced by sAd4287vectors bearing SIVmac239 Gag in C57BL/6 mice immunized with 10⁷, 10⁸,and 10⁹ viral particles (vp) of the vector, as assessed by measuring theCD8⁺ T cell response via D^(b)/AL11 tetramer binding assays at days 0,7, 14, 21, and 28 post-immunization.

FIG. 23B is a graph showing the cellular responses induced by sAd4310Avectors bearing SIVmac239 Gag in C57BL/6 mice immunized with 10⁷, 10⁸,and 10⁹ viral particles (vp) of the vector, as assessed by measuring theCD8⁺ T cell response via D^(b)/AL11 tetramer binding assays at days 0,7, 14, 21, and 28 post-immunization.

FIG. 24A is a graph showing the cellular responses induced by sAd4287,sAd4310A, and replication-competent sAd4287 (rcsAd4287) at 10⁹ vp asdetermined by IFN-γ ELISPOT assays using splenocytes from C57BL/6 miceon day 28 post-immunization. IFN-γ ELISPOT responses were measured tooverlapping Gag peptides (Gag), the dominant CD8⁺ T cell epitope AL11,the sub-dominant CD8⁺ epitope KV9, and the CD4⁺ T cell epitope DD13.

FIG. 24B is a graph showing the cellular responses induced by sAd4287,sAd4310A, and rcsAd4287 at 10⁸ vp as determined by IFN-γ ELISPOT assaysusing splenocytes from C57BL/6 mice on day 28 post-immunization. IFN-γELISPOT responses were measured to Gag, the dominant CD8⁺ T cell epitopeAL11, the sub-dominant CD8⁺ T epitope KV9, and the CD4⁺ T cell epitopeDD13.

FIG. 24C is a graph showing the cellular responses induced by sAd4287,sAd4310A, and rcsAd4287 at 10⁷ vp as determined by IFN-γ ELISPOT assaysusing splenocytes from C57BL/6 mice on day 28 post-immunization. IFN-γELISPOT responses were measured to Gag, the dominant CD8⁺ T cell epitopeAL11, the sub-dominant CD8⁺ T epitope KV9, and the CD4⁺ T cell epitopeDD13.

DETAILED DESCRIPTION

We have previously identified a variety of novel viruses, includingseveral novel adenoviruses, from rhesus monkeys as part of ametagenomics study (Handley et al. Cell, 151(2):253-266, 2012). In thepresent invention, we isolated, amplified, and purified three novelsimian adenoviruses (sAds), sAd4287, sAd4310 #13-1 (sAd4310A), andsAd4312. The three skis were obtained from the rhesus monkeymetagenomics study described above. These viruses are entirely novel andtheir full sequences have never previously peen reported. As theseviruses have not yet been officially “named,” they do not yet have anofficial adenovirus number. Accordingly, the nomenclature usedthroughout represents our internal laboratory designation.

The complete genome sequence of the novel sAds as well as the vectorsystems we generated for each of the viruses is described in detailbelow. We generated recombinant sAd4287, sAd4310A, and sAd4312 vectorsexpressing a variety of transgenes, including luciferase and SIV Gag. Inaddition, we demonstrated that these vectors (i) have extremely andsurprisingly low seroprevalence in human populations and (ii) exhibitpotent immunogenicity in mice. This combination of low baselineanti-vector immunity and potent immunogenicity suggests that these noveladenoviral vectors can be useful in the generation of vaccines againstdiseases, such as cancer and those caused by an infective agent.

Polynucleotides of the Invention

As a first aspect, the invention provides polynucleotide sequencesrelated to the three novel sAds (sAd4287, sAd4310A, and sAd4312). Theisolated polynucleotides may include a nucleotide sequence that is atleast 90% identical (e.g., at least 91%, 92%, 93%, or 94% identical), atleast 95% identical (e.g., at least 96%, 97%, 98%, or 99% identical), or100% identical to all or a portion of any one of the full-length genomesequence of wild-type sAd4287 (SEQ ID NO: 1), sAd4310A (SEQ ID NO: 2),or sAd4312 (SEQ ID NO: 3), or their complement. The isolatedpolynucleotides of the invention may include at least 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000,20000, 25000, 30000, 35000 or more contiguous or non-contiguousnucleotides of SEQ ID NOs: 1-3.

In some embodiments, the polynucleotides of the invention may be used asprimers that are between 10-100 nucleotides in length, more particularlybetween 10-30 nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides inlength), and can be at least 90% identical (e.g., at least 91%, 92%,93%, or 94% identical), at least 95% identical (e.g., at least 96%, 97%,98%, or 99% identical), or 100% identical to any one of SEQ ID NOs:52-123.

In some embodiments, the polynucleotides of the invention include all ora portion of the nucleotide sequence encoding the fiber-1, fiber-2;and/or hexon protein of wild-type sAd4287, sAd4310A, and/or sAd4312. Insome embodiments, the nucleotide sequence encoding all or a portion ofthe fiber-1 protein can be at least 90% identical (e.g., at least 91%,92%, 93%, or 94% identical), at least 95% identical (e.g., at least 96%,97%, 98%, or 99% identical), or 100% identical to the nucleotidesequence encoding the fiber-1 protein of wild-type sAd4287, sAd4310A, orsAd4312, which corresponds to SEQ ID NO: 4, 5, and 6, respectively. Thepolypeptide sequences of the fiber-1 protein of wild-type sAd4287,sAd4310A, and sAd4312 correspond to SEQ ID NOs: 19, 20, and 21,respectively. In some embodiments, the nucleotide sequence encoding allor a portion of the fiber-2 protein can be at least 90% identical (e.g.,at least 91%, 92%, 93%, or 94% identical), at least 95% identical (e.g.,at least 96%, 97%, 98%, or 99% identical), or 100% identical to thenucleotide sequence encoding the fiber-2 protein of wild-type sAd4287,sAd4310A, or sAd4312, which corresponds to SEQ ID NO: 7, 8, and 9,respectively. The polypeptide sequences of the fiber-2 protein ofwild-type sAd4287, sAd4310A, and sAd4312 correspond to SEQ ID NOs: 22,23, and 24, respectively. In some embodiments, the nucleotide sequenceencoding all or a portion of the hexon protein can be at least 90%identical (e.g., at least 91%, 92%, 93%, or 94% identical), at least 95%identical (e.g., at least 96%, 97%, 98%, or 99% identical), or 100%identical to the nucleotide sequence encoding the hexon protein ofwild-type sAd4287, sAd4310A, or sAd4312, which corresponds to SEQ ID NO:10, 11, and 12, respectively. The polypeptide sequences of the hexonprotein of wild-type sAd4287, sAd4310A, and sAd4312 correspond to SEQ IDNOs: 25, 26, and 27, respectively.

In other embodiments, the polynucleotides of the invention include allor a portion of the nucleotide sequence encoding the knob domain offiber-1 of wild-type sAd4287, sAd4310A, and/or sAd4312. In someembodiments, the nucleotide sequence encoding all or a portion of theknob domain of fiber-1 can be at least 90% identical (e.g., at least91%, 92%, 93%, or 94% identical), at least 95% identical (e.g., at least96%, 97%, 98%, or 99% identical), or 100% identical to the nucleotidesequence encoding the knob domain of the fiber-1 protein of wild-typesAd4287, sAd4310A, or sAd4312, which corresponds to SEQ ID NO: 13, 14,or 15, respectively. The polypeptide sequences of the knob domain of thefiber-1 protein of wild-type sAd4287, sAd4310A, and sAd4312 correspondto SEQ ID NOs: 28, 29, and 30, respectively. In some embodiments, thenucleotide sequence encoding all or a portion of the knob domain offiber-2 can be at least 90% identical (e.g., at least 91%, 92%, 93%, or94% identical), at least 95% identical (e.g., at least 96%, 97%, 98%, or99% identical), or 100% identical to the nucleotide sequence encodingthe knob domain of the fiber-2 protein of wild-type sAd4287, sAd4310A,or sAd4312, which corresponds to SEQ ID NO: 16, 17, and 18,respectively. The polypeptide sequences of the knob domain of thefiber-2 protein of wild-type sAd4287, sAd4310A, and sAd4312 correspondto SEQ ID NOs: 31, 32, and 33, respectively.

In other embodiments, the polynucleotides of the invention include allor a portion of one or more of the nucleotide sequences encoding thefiber-1, fiber-2, hexon, fiber-1 knob, and/or fiber-2 knob proteins ofsAd4287, sAd4310A, and/or sAd4312 and nucleotide sequence from one ormore adenoviral vectors including Ad11, Ad15, Ad24, Ad26, Ad34, Ad35,Ad48, Ad49, Ad50, and/or Pan9 (also known as AdC68) directed to thegeneration of chimeric adenoviral vectors, as discussed below. In otherembodiments, the polynucleotides of the invention include all or aportion of one or more of the nucleotide sequences encoding the fiber-1,fiber-2, hexon, fiber-1 knob, and/or fiber-2 knob proteins of sAd4287,sAd4310A, and/or sAd4312 and nucleotide sequence that can be at least90% identical (e.g., at least 91%, 92%, 93%, or 94% identical), at least95% identical (e.g., at least 96%, 97%, 98%, or 99% identical), or 100%identical to nucleotide sequence from one or more adenoviral vectorsincluding Ad11, Ad15, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49, Ad50, and/orPan9 (also known as AdC68). In other embodiments, the polynucleotides ofthe invention include nucleotide sequence from one or more adenoviralvectors including Ad5, Ad11, Ad15, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49,Ad50, and/or Pan9 (also known as AdC68) and all or a portion of one ormore of a nucleotide sequence that can be at least 90% identical (e.g.,at least 91%, 92%, 93%, or 94% identical), at least 95% identical (e.g.,at least 96%, 97%, 98%, or 99% identical), or 100% identical to all or aportion of one or more of the nucleotide sequences encoding the fiber-1,fiber-2, hexon, fiber-1 knob, and/or fiber-2 knob proteins of sAd4287,sAd4310A, and/or sAd4312.

Vectors of the Invention

The present invention also features recombinant vectors including anyone or more of the polynucleotides described above. In some embodiments,one vector of the invention can be used in conjunction with one or moreother vectors (e.g., 1, 2, 3, or more vectors) of the invention as avector system, which can be used to generate recombinantreplication-defective sAds (rdsAds) or replication-competent sAds(rcsAds) of the invention. Accordingly, the invention features noveladenovirus vector systems for each of the three novel sAds (sAd4287,sAd4310A, and sAd4312) described herein. Such vector systems to generatereplication-defective adenoviruses are known in the art and have beenapplied to generate replication competent adenovirus-free batches basedof, for example, Ad5, Ad11, Ad35 and Ad49 (see, e.g., WO 97/00326, WO00/70071; WO 02/40665; U.S. Pub. No. 2005/0232900, all incorporatedherein by reference). However, the vectors and vector systems of thepresent invention, applied towards the sAds sAd4287, sAd4310A, andsAd4312 are novel.

In some embodiments, the vectors of the invention can contain the E1region (e.g., nt 474 to nt 3065 of sAd4287 (SEQ ID NO: 1); nt 474 to nt3088 of sAd4310A (SEQ ID NO: 2); and nt 487 to nt 3100 of sAd4312 (SEQID NO: 3)) of the specific sAd (e.g., sAd4287, sAd4310A, and sAd4312)for the purposes of producing replication-competent sAd (rcsAd). Suchvectors are exemplified, for example, in the .E1btg.Empty vectors of theinvention (see, e.g., FIGS. 7, 14, and 21, which depict the .E1btg.Emptyvectors of the invention for each of the three novel adenoviruses).

In some embodiments, the vectors of the invention can contain theleft-end sAd sequences and an expression/transgene cassette (see, e.g.,FIG. 3, depicting the pBr/sAd4287.pIX-pV vector that includes the leftpart of the sAd4287 genome from approximately pIX to pV). In someembodiments, the expression cassette of the vector replaces or disruptsthe E1 region of the specific adenovirus. In preferred embodiments, theexpression cassette includes a promoter (e.g., a CMV promoter, e.g., aCMVlong promoter) that stimulates expression of a transgene, andoptionally a poly-adenylation signal (e.g., a heterologous nucleotidesequence encoding an antigenic gene product of interest, e.g., abacterial, viral, parasitic, fungal, or therapeutic protein, or fragmentthereof) (see, e.g., FIGS. 2, 9, and 16, depicting .Empty vectors of theinvention for each of the three novel adenoviruses). The E1 region canbe deleted (either partially or completely), disrupted, or renderedinactive, by one or more mutations.

In some embodiments, the vectors of the invention can contain the leftpart of the sAd sequences (see, e.g., FIG. 3, depicting thepBr/sAd4287.pIX-pV vector that includes the left part of the sAd4287genome from approximately pIX to pV), which includes the penton base and52 K coding regions of the sAd (see, e.g., FIGS. 3, 10, and 17,depicting the .pIX-pV vectors of the invention for each of the threenovel adenoviruses).

In other embodiments, the vectors of the invention can contain the rightpart of the sAd sequences (see, e.g., FIG. 4, depicting thepBr/sAd4287,PsiI.rITR vector that includes the right part of the sAd4287genome from approximately pVII to the right ITR (rITR)) (see, e.g.,FIGS. 4, 11, and 18, depicting the .pV-rITR vectors of the invention foreach of the three novel adenoviruses). In some embodiments, thesevectors may further have a deleted, disrupted, or mutated E3 (e.g., nt25973 to nt 28596 of sAd4287 (SEQ ID NO: 1); nt 25915 to nt 28496 ofsAd4310A (SEQ ID NO: 2): and nt 25947 to nt 28561 of sAd4312 (SEQ ID NO:3); see FIGS. 5, 12, and 19, depicting the .dE3 vectors of the inventionfor each of the three novel adenoviruses) and/or E4 region (e.g., nt31652 to nt 34752 of sAd4267 (SEQ ID NO: 1); nt 31750 to nt 34048 ofsAd4310A (SEQ ID NO: 2); and nt 31818 to nt 34116 of sAd4312 (SEQ ID NO:3); see FIGS. 6, 13, and 20, depicting the .dE3.dE4 vectors of theinvention for each of the three novel adenoviruses), which are notrequired for replication and packaging of the adenoviral particle.Deletion of the E3 region is generally preferred if large transgenesequences are to be incorporated into the vector since the genome sizewhich can be packaged into a functional particle is limited toapproximately 105% of the wild type size. Although not applied herein,it is to be understood that other modifications may be introduced in theadenoviral genome, such as deletion of the E2A region, or most if notall of the entire E4 region. In some embodiments, a cell transfectedwith a vector of the invention can complement these deficiencies bydelivering the functionality of the missing regions. The E2A region canbe provided by, for instance, a temperature sensitive E2A mutant, or bydelivering the E4 functions. Cells that can be used to complement adeficiency of an adenoviral gene (e.g., a E1, E3, and/or E4 deletion) ofa vector of the invention include, for example, PER.55K, PER.C6®, and293 cells. All such systems are known in the art and such modificationsof the adenoviral genomes are within the scope of the present invention,which in principal relates to the three novel sAd4287, sAd4310A, andsAd4312 genomic sequences, and the use thereof. As described above, anyone vector of the invention can be used in conjunction with one or moreother vectors of the invention. In some embodiments, vectors are usedwhich encode both left and right sides of the sAd genome in order togenerate a given sAd of the invention.

The present invention also features vectors for the generation ofchimeric adenoviruses which include a portion of the sAd4287, sAd4310A,or sAd4312 genome as well as a portion of the genome of one or moreother viruses. In some embodiments, the chimeric adenoviral vectors ofthe invention may include a substitution of all or a portion of thehexon and/or fiber protein. In some embodiments, the portion of thehexon protein substituted with that of another virus is one or more ofthe hexon protein hypervariable regions (HVRs), for example, HVR1 (nt403 to nt 489), HVR2 (nt 520 to nt 537), HVR3 (nt 592 to nt 618), HVR4(nt 706 to nt 744), HVR5 (nt 763 to 786), HVR6 (nt 856 to nt 874),and/or HVR7 nt 1201 to nt 1296) of sAd4287 hexon protein (SEQ ID NO:10); HVR1 (nt 403 to nt 477), HVR2 (nt 505 to nt 516), HVR3 (nt 571 tont 591), HVR4 (nt 679 to nt 690), HVR5 (nt 709 to 735), HVR6 (nt 805 tont 816), and/or HVR7 (nt 1144 to nt 1236) of sAd4310A hexon protein (SEQID NO: 11); or HVR1 (nt 403 to nt 474), HVR2 (nt 505 to nt 522), HVR3(nt 577 to nt 597), HVR4 (nt 685 to nt 726), HVR5 (nt 748 to 777), HVR6(nt 847 to nt 864), and/or HVR7 (nt 1192 to nt 1284) of sAd4312 hexonprotein (SEQ ID NO: 12). In some embodiments, the portion of the fiberprotein substituted with that of another virus is the fiber knob domain.In some embodiments, the substituted regions are replaced with a regionderived from an adenovirus that has a lower seroprevalence compared tothat of Ad5, such as subgroup B (Ad11, Ad34, Ad35, and Ad50) andsubgroup D (Ad15, Ad24, Ad26, Ad48, and Ad49) adenoviruses as well assimian adenoviruses (e.g., Pan9, also known as AdC68). In someembodiments, an adenoviral vector backbone of Ad5, Ad11, Ad15, Ad24,Ad26, Ad34, Ad48, Ad49, Ad50, or Pan9/AdC68 includes a substitution ofall or a portion of one or more of the above hexon HVRs of sAd4287,sAd4310A, and/or sAd4312.

Adenoviruses of the Invention

As discussed above, a recombinant adenovirus of the invention derived,at least in part, from sAd4287, sAd4310A, and/or sAd4312 can begenerated using the above-described vectors of the invention. Theseadenoviruses may be rcsAds or rdsAds. rdsAds will include a deleted,disrupted, or mutational inactivation of the E1 region, and may furtherinclude a deletion, disruption, or mutational inactivation of the E2,E3, and/or E4 regions. In some embodiments, the adenovirus of theinvention may include an antigenic or therapeutic gene product, orfragment thereof, including a bacterial, viral, parasitic, or fungalprotein, or fragment thereof. In a preferred embodiment, the antigenicgene product, or fragment thereof, when expressed in a host, or hostcells, is capable of eliciting a strong immune response. In someembodiments, the bacterial protein, or fragment thereof, may be derivedfrom Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacteriumafricanum, Mycobacterium microti, Mycobacterium leprae, Pseudomonasaeruginosa, Salmonella typhimurium, Escherichia coil, Klebsiellapneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, Francisellatularensis, Brucella, Burkholderia mallei, Yersinia pestis,Corynebacterium Neisseria meningitidis, Bordetella pertussis,Clostridium tetani, or Bacillus anthracis. In some embodiments, theviral protein, or fragment thereof, may be derived from a virus of aviral family selected from the group consisting of Retroviridae,Flaviviridae, Arenaviridae, Bunyaviridae, Filoviridae, Togaviridae,Poxviridae, Herpesviridae, Orthomyxoviridae, Coronaviridae,Rhabdoviridae, Paramyxoviridae, Picornaviridae, Hepadnaviridae,Papillomaviridae, Parvoviridae, Astroviridae, Polyomaviridae,Calciviridae, and Reoviridae. In some embodiments, the virus is humanimmunodeficiency virus (HIV), human papillomavirus (HPV), hepatitis Avirus (Hep A), hepatitis B virus (HBV), hepatitis C virus (HCV), Variolamajor, Variola minor, monkeypox virus, measles virus, rubella virus,mumps virus, varicella zoster virus (VZV), poliovirus, rabies virus,Japanese encephalitis virus, herpes simplex virus (HSV), cytomegalovirus(CMV), rotavirus, influenza, Ebola virus, yellow fever virus, or Marburgvirus. In some embodiments, the parasitic protein, or fragment thereof,is from Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax,Plasmodium ovale, Plasmodium malariae, Trypanosome spp., or Legionellaspp. In some embodiments, the fungal protein, or fragment thereof, isfrom Aspergillus, Blastomyces dermatitidis, Candida, Coccidioidesimmitis, Cryptococcus neoformans, Histoplasma capsulatum var.capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii,Zygomycetes spp., Absidia corymbifera, Rhizomucor pusillus, or Rhizopusarrhizus. In some embodiments, the therapeutic gene products may beinterferon (IFN) proteins, Factor VIII, Factor IX, erythropoietin,alpha-1 antitrypsin, calcitonin, glucocerebrosidase, growth hormone, lowdensity lipoprotein (LDL), receptor IL-2 receptor and its antagonists,insulin, globin, immunoglobulins, catalytic antibodies, theinterleukins, insulin-like growth factors, superoxide dismutase, immuneresponder modifiers, parathyroid hormone and interferon, nerve growthfactors, tissue plasminogen activators, and/or colony stimulatingfactors (see, e.g., U.S. Pat. No. 6,054,288, incorporated by referenceherein). In some embodiments, the IFN protein has an amino acid sequencesubstantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or even 100% identical) to the sequence of a humanIFN-α (e.g., IFN-α-1α, IFN-α-1b, IFN-α-2α, IFN-α-2b, and consensus IFN-α(conIFN-α); FIG. 1), a human IFN-β (e.g., IFN-β-1a and IFN-β-1b), ahuman IFN-γ), or an IFN-τ or a polypeptide that demonstrates the same orsimilar biological activity to an interferon (e.g., at least 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, or 100% of the activity of a human IFN-α,a human IFN-β, a human IFN-γ, an IFN-τ, or a conIFN-α (see, e.g., U.S.Pat. No. 4,695,623 and U.S. Pub. No. 2011/0000480, incorporated byreference herein, for examples of specific IFN sequences).

Non-limiting examples of bacterial gene products, or fragments thereof,include 10.4, 85A, 85B, 86C, CFP-10, Rv3871, and ESAT-6 gene products,or fragments thereof, of Mycobacterium, O, H, and K antigens, orfragments thereof, of E. coli; and protective antigen (PA), or fragmentsthereof, of Bacillus anthracis. Non-limiting examples of viral geneproducts, or fragments thereof, include Gag, Pol, Nef, Tat, Rev, Vif,Vpr, or Vpu, or fragments thereof, of HIV and other retroviruses (see,e.g., U.S. Pub. No. 2012/0076812, incorporated by reference herein); 9Dantigen, or fragments thereof, of HSV; Env, or fragments thereof, of allenvelope protein-containing viruses. Non-limiting examples of parasiticgene products, or fragments thereof, include circumsporozoite (CS)protein, gamete surface proteins Pfs230 and Pfs48/45, and Liver SpecificAntigens 1 or 3 (LSA-1 or LSA-3), or fragments thereof, of Plasmodiumfalciparum. Non-limiting examples of fungal gene products, or fragmentsthereof, include any cell wall mannoprotein (e.g., Afmp1 of Aspergillusfumigatus) or surface-expressed glycoprotein (e.g., SOWgp ofCoccidioides immitis).

Methods of Prophylaxis or Treatment Using Compositions of the Invention

The pharmaceutical compositions of the invention can be used as vaccinesfor treating a subject (e.g., a human) with a disease (e.g., cancer or adisease caused by an infective agent, e.g., AIDS). In particular, thecompositions of the invention car be used to treat (pre- orpost-exposure) infection by bacteria, including Mycobacteriumtuberculosis, Mycobacterium bovis, Mycobacterium africanum,Mycobacterium microti, Mycobacterium leprae, Pseudomonas aeruginosa,Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae,Streptococcus pneumoniae, Staphylococcus aureus, Francisella tularensis,Brucella, Burkholderia mallei, Yersinia pestis, Corynebacteriumdiphtheria, Neisseria meningitidis, Bordetella pertussis, Clostridiumtetani, or Bacillus anthracis; viruses of a viral family selected fromthe group consisting of Retroviridae, Flaviviridae, Arenaviridae,Bunyaviridae, Filoviridae, Togaviridae, Poxviridae, Herpesviridae,Orthomyxoviridae, Coronaviridae, Rhabdoviridae, Paramyxoviridae,Picornaviridae, Hepadnaviridae, Papillomaviridae, Parvoviridae,Astroviridae, Polyomaviridae, Calciviridae, and Reoviridae; parasites,including Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax,Plasmodium ovale, Plasmodium malariae, Trypanosoma spp., or Legionellaspp.; and fungi, including Aspergillus, Blastomyces dermatitidis,Candida, Coccidioides immitis, Cryptococcus neoformans, Histoplasmacapsulatum var. capsulatum, Paracoccidioides brasiliensis, Sporothrixschenckii, Zygomycetes spp., Absidia corymbifera, Rhizomucor pusillus,or Rhizopus arrhizus.

Accordingly, in other non-limiting embodiments, the pharmaceuticalcompositions of the invention can be used to treat a subject (e.g., ahuman) with acquired immune deficiency syndrome (AIDS), cancer,tuberculosis, leprosy, typhoid fever, pneumonia, meningitis,staphylococcal scalded skin syndrome (SSSS), Ritter's disease, tularemia(rabbit fever), brucellosis, Glanders disease, bubonic plague,septicemic plague, pneumonic plague, diphtheria, pertussis (whoopingcough), tetanus, anthrax, hepatitis, smallpox, monkeypox, measles,mumps, rubella, chicken pox, polio, rabies, Japanese encephalitis,herpes, mononucleosis, influenza, Ebola virus disease, hemorrhagicfever, yellow fever, Marburg virus disease, toxoplasmosis, malaria,trypanosomiasis, legionellosis, aspergillosis, blastomycosis,candidiasis (thrush), coccidioidomycosis, cryptococcosis,histoplasmosis, paracoccidioidomycosis, sporotrichosis, or sinus-orbitalzygomycosis.

Pharmaceutical Formulation and Administration of the Compositions of theInvention Administration

The pharmaceutical compositions of the invention can be administered toa subject (e.g., a human), pre- or post-exposure to an infective agent(e.g., bacteria, viruses, parasites, fungi) or pre- or post-diagnosis ofa disease of a disease without an etiology traceable to an infective,agent (e.g., cancer), to treat, prevent, ameliorate, inhibit theprogression of, or reduce the severity of one or more symptoms of thedisease in the subject. For example, the compositions of the inventioncan be administered to a subject to treat having AIDS. Examples ofsymptoms of diseases caused by a viral infection, such as AIDS, that canbe treated using the compositions of the invention include, for example,fever, muscle aches, coughing, sneezing, runny nose, sore throat,headache, chills, diarrhea, vomiting, rash, weakness, dizziness,bleeding under the skin, in internal organs, or from body orifices likethe mouth, eyes, or ears, shock, nervous system malfunction, delirium,seizures, renal (kidney) failure, personality changes, neck stiffness,dehydration, seizures, lethargy, paralysis of the limbs, confusion, backpain, loss of sensation, impaired bladder and bowel function, andsleepiness that can progress into coma or death. These symptoms, andtheir resolution during treatment, may be measured by, for example, aphysician during a physical examination or by other tests and methodsknown in the art.

The compositions utilized in the methods described herein can beformulated, for example, for administration intramuscularly,intravenously, intradermally, percutaneously, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrapleurally, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, topically,intratumorally, peritoneally, subcutaneously, subconjunctivally,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularly, orally, topically, locally, by inhalation, by injection,by infusion, by continuous infusion, by localized perfusion bathingtarget cells directly, by catheter, by lavage, by gavage, in cremes, orin lipid compositions.

The preferred method of administration can vary depending on variousfactors (e.g., the components of the composition being administered andthe severity of the condition being treated). Formulations suitable fororal or nasal administration may consist of liquid solutions, such as aneffective amount of the composition dissolved in a diluent (e.g., water,saline, or PEG-400), capsules, sachets, tablets, or gels, eachcontaining a predetermined amount of the chimeric Ad5 vector compositionof the invention. The pharmaceutical composition may also be an aerosolformulation for inhalation, for example, to the bronchial passageways.Aerosol formulations may be mixed with pressurized, pharmaceuticallyacceptable propellants (e.g., dichlorodifluoromethane, propane, ornitrogen). In particular, administration by inhalation can beaccomplished by using, for example, an aerosol containing sorbitantrioleate or oleic acid, for example, together withtrichlorofluoromethane, dichlorofluoromethane,dichlorotetrafluoroethane, or any other biologically compatiblepropellant gas.

Immunogenicity of the composition of the invention may be significantlyimproved if it is co-administered with an immunostimulatory agent oradjuvant. Suitable adjuvants well-known to those skilled in the artinclude, for example, aluminum phosphate, aluminum hydroxide, QS21, QuilA (and derivatives and components thereof), calcium phosphate, calciumhydroxide, zinc hydroxide, glycolipid analogs, octodecyl esters of anamino acid, muramyl dipeptides, polyphosphazene, lipoproteins, ISCOMmatrix, DC-Chol, DDA, cytokines, and other adjuvants and derivativesthereof.

Pharmaceutical compositions according to the invention described hereinmay be formulated to release the composition immediately uponadministration (e.g., targeted delivery) or at any predetermined timeperiod after administration using controlled or extended releaseformulations. Administration of the pharmaceutical composition incontrolled or extended release formulations is useful where thecomposition, either alone or in combination, has (i) a narrowtherapeutic index (e.g., the difference between the plasma concentrationleading to harmful side effects or toxic reactions and the plasmaconcentration leading to a therapeutic effect is small; generally, thetherapeutic index, TI, is defined as the ratio of median lethal dose(LD₅₀) to median effective dose (ED₅₀)); (ii) a narrow absorption windowat the site of release (e.g., the gastro-intestinal tract); or (iii) ashort biological half-life, so that frequent dosing during a day isrequired in order to sustain a therapeutic level.

Many strategies can be pursued to obtain controlled or extended releasein which the rate of release outweighs the rate of metabolism of thepharmaceutical composition. For example, controlled release can beobtained by the appropriate selection of formulation parameters andingredients, including, e.g., appropriate controlled releasecompositions and coatings. Suitable formulations are known to those ofskill in the art. Examples include single or multiple unit tablet orcapsule compositions, oil solutions, suspensions, emulsions,microcapsules, microspheres, nanoparticles, patches, and liposomes.

The compositions of the invention may be administered to providepre-exposure prophylaxis or after a subject has been diagnosed with adisease having a disease without an etiology traceable to an infectiveagent (e.g., cancer) or a subject exposed to an infective agent, such asa bacterium, virus, parasite, or fungus. The composition may beadministered, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,35, 40, 45, 50, 55, or 60 minutes, 2, 4, 6, 10, 15, or 24 hours, 2, 3,5, or 7 days, 2, 4, 6 or 8 weeks, or even 3, 4, or 6 months pre-exposureor pre-diagnosis, or may be administered to the subject 15-30 minutes or1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 20, 24, 48, or 72 hours, 2, 3, 5, or7 days, 2, 4, 6 or 8 weeks, 3, 4, 6, or 9 months, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, or 20 years or longer post-diagnosis or post-exposure tothe infective agent.

When treating disease (e.g., AIDS or cancer), the compositions of theinvention may be administered to the subject either before theoccurrence of symptoms or a definitive diagnosis or after diagnosis orsymptoms become evident. For example, the composition may beadministered, for example, immediately after diagnosis or the clinicalrecognition of symptoms or 2, 4, 6, 10, 15, or 24 hours, 2, 3, 5, or 7days, 2, 4, 6 or 8 weeks, or even 3, 4, or 6 months after diagnosis ordetection of symptoms.

The compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation may be administered in powder form or combined with asterile aqueous carrier prior to administration. The pH of thepreparations typically will be between 3 and 11, more preferably between5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as7 to 7.5. The resulting compositions in solid form may be packaged inmultiple single dose units, each containing a fixed amount of therecombinant replication-defective sAd vector containing a heterologousnucleic acid encoding an antigenic gene product, or fragment thereof,(e.g., an sAd4287, sAd4310A, or sAd4312 HIV Gag delivery vector) and, ifdesired, one or more immunomodulatory agents, such as in a sealedpackage of tablets or capsules, or in a suitable dry powder inhaler(DPI) capable of administering one or more doses.

Dosages

The dose of the compositions of the invention (e.g., the number ofantigenic gene product-encoding recombinant sAd vectors) or the numberof treatments using the compositions of the invention may be increasedor decreased based on the severity of, occurrence of, or progression of,the disease in the subject (e.g., based on the severity of one or moresymptoms of, e.g., viral infection or cancer).

The pharmaceutical compositions of the invention can be administered ina therapeutically effective amount that provides an immunogenic and/orprotective effect against an infective agent or target protein for adisease caused by a non-infective agent. For example, the subject can beadministered at least about 1×10³ viral particles (vp)/dose or between1×10¹ and 1×10¹⁴ vp/dose, preferably between 1×10³ and 1×10¹² vp/dose,and more preferably between 1×10⁵ and 1×10¹¹ vp/dose.

Viral particles include nucleic acid molecules encoding an antigenicgene product or fragment thereof (e.g., viral structural andnon-structural proteins) and are surrounded by a protective coat (aprotein-based capsid with hexon and fiber proteins, which may be derivedfrom a single sAd of the invention or a chimeric variant thereof). Viralparticle number can be measured based on, for example, lysis of vectorparticles, followed by measurement of the absorbance at 260 nm (see,e.g., Steel, Curr. Opin. Biotech., 1999).

The dosage administered depends on the subject to be treated (e.g., theage, body weight, capacity of the immune system, and general health ofthe subject being treated), the form of administration (e.g., as a solidor liquid), the manner of administration (e.g., by injection,inhalation, dry powder propellant), and the cells targeted (e.g.,epithelial cells, such as blood vessel epithelial cells, nasalepithelial cells, or pulmonary epithelial cells). The composition ispreferably administered in an amount that provides a sufficient level ofthe antigenic or therapeutic gene product, or fragment thereof (e.g., alevel of an antigenic gene product that elicits an immune responsewithout undue adverse physiological effects in the host caused by theantigenic gene product).

In addition, single or multiple administrations of the compositions ofthe present invention may be given (pre- or post-exposure and/or pre- orpost-diagnosis) to a subject (e.g., one administration or administrationtwo or more times). For example, subjects who are particularlysusceptible to, for example, viral infection may require multipletreatments to establish and/or maintain protection against the virus.Levels of induced immunity provided by the pharmaceutical compositionsdescribed herein can be monitored by, for example, measuring amounts ofneutralizing secretory and serum antibodies. The dosages may then beadjusted or repeated as necessary to trigger the desired level of immuneresponse. For example, the immune response triggered by a singleadministration (prime) of a composition of the invention may notsufficiently potent and/or persistent to provide effective protection.Accordingly, in some embodiments, repeated administration (boost), suchthat a prime boost regimen is established, can significantly enhancehumoral and cellular responses to the antigen of the composition.

Alternatively, the efficacy of treatment can be determined by monitoringthe level of the antigenic or therapeutic gene product, or fragmentthereof, expressed in a subject (e.g., a human) following administrationof the compositions of the invention. For example, the blood or lymph ofa subject can be tested for antigenic or therapeutic gene product, orfragment thereof, using, for example, standard assays known in the art(see, e.g., Human Interferon-Alpha Multi-Species ELISA kit (Product No.41105) and the Human Interferon-Alpha Serum Sample kit (Product No.41110) from Pestka Biomedical Laboratories (PBL), Piscataway, N.J.).

A single dose of the compositions of the invention may achieveprotection, pre exposure or pre-diagnosis. In addition, a single doseadministered post-exposure or post-diagnosis can function as a treatmentaccording to the present invention.

A single dose of the compositions of the invention can also be used toachieve therapy in subjects being treated for a disease. Multiple doses(e.g., 2, 3, 4, 5, or more doses) can also be administered, innecessary, to these subjects.

Carriers, Excipients, Diluents

The compositions of the invention include sAd5 vectors containing aheterologous nucleic acid molecule encoding an antigenic or therapeuticgene product, or fragment thereof. Therapeutic formulations of thecompositions of the invention are prepared using standard methods knownin the art by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences (20^(th) edition), ed.A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).Acceptable carriers, include saline, or buffers such as phosphate,citrate and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilicpolymers such as polyvinylpyrrolidone, amino acids such as glycine,glutamine, asparagines, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN™, PLURONICS™, or PEG.

Optionally, but preferably, the formulation contains a pharmaceuticallyacceptable salt, preferably sodium chloride, and preferably at aboutphysiological concentrations. Optionally, the formulations of theinvention can contain a pharmaceutically acceptable preservative. Insome embodiments the preservative concentration ranges from 0.1 to 2.0%,typically v/v. Suitable preservatives include those known in thepharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben,and propylparaben are preferred preservatives. Optionally, theformulations of the invention can include a pharmaceutically acceptablesurfactant at a concentration of 0.005 to 0.02%.

EXAMPLES

The following examples are to illustrate the invention. They are notmeant to limit the invention in any way.

The practice of this invention may employ, unless otherwise indicated,conventional techniques of molecular biology, cell biology, andrecombinant DNA, which are within the skill of the person skilled in theart (see, e.g., Green and Sambrook. Molecular Cloning: A LaboratoryManuel, 4^(th) edition, 2012; Ausubel, et al. Current Protocols inMolecular Biology, 1987; Methods in Enzymology. Academic Press, Inc.;and MacPherson et al. PCR2: A Practical Approach, 1995).

Example 1. Sequence of Simian Adenovirus sAd4287

The total genome sequence of simian adenovirus sAd4287 was determinedfollowing the isolation, amplification, and purification of the novelvirus obtained from the rhesus monkey metagenomics study of Handley etal. (Cell. 151(2):253-266, 2012). The obtained sequence of the sAd4287genome (35079 nucleotides (nt)) is given as SEQ ID NO: 1. A schematicgenome structure of sAd4287 is depicted in FIG. 1. Using the fullgenomic sequence in an NCBI web-based BLAST search, the most closelyrelated virus to sAd4287 was identified as simian adenovirus 1 (sAd1)ATCC VR-195 (query coverage: 93%; maximum identity: 98%). NCBI web-basedBLAST searches were also performed to assess homology of three majorcapsid proteins of sAd4287 (fiber-1, fiber-2, and hexon proteins). Themost closely related protein to sAd4287 fiber-1 was identified as sAd1fiber-1 (query coverage: 100%; maximum identity: 74%). The most closelyrelated protein to sAd4287 fiber-2 was identified as sAd7 long fiber(query coverage: 100%; maximum identity: 97%). The most closely relatedprotein to sAd4287 hexon was identified as sAd1 hexon (query coverage:100%; maximum identity: 93%).

Example 2. Generation of Recombinant sAd4287 Viruses

Here, the construction of an sAd4287 plasmid-based system to generaterecombinant sAd4287 vectors in a safe and efficient manner is described.The plasmid system consists of a first plasmid, referred to as anadapter plasmid, which contains sAd4287 nucleotides 1 to 460 includingthe left inverted terminal repeat (lITR) and packaging signal, anexpression cassette and an sAd4287 fragment corresponding to nucleotides2966 to 5466. The expression cassette comprises the human CMV promoter,a multiple cloning site (MCS), and the SV40 polyadenylation signal(polyA) as previously described (see, e.g., WO 00/70071). The adapterplasmid is based on pAdApt26.Empty (Abbink, et al, J. Virol. 81(9):4654-4663, 2007), albeit now generated to comprise the sAd4287-derivedsequences instead of the Ad26-derived sequences. Furthermore, the systemconsists of other plasmids together constituting sAd4287 sequencesbetween nucleotide 2966 and 35079 that may be deleted for E1 region (nt474 to nt 3085 of SEQ ID NO: 1), E3 region (nt 25973 to nt 28596 of SEQID NO: 1), and/or E4 region (nt 31852 to nt 34752 of SEQ ID NO: 1)sequences.

Generation of Adapter Plasmid sAdApt4287.Empty

Plasmids that were used for harboring the sAd4287 sequences wereprepared. Primers (sAd4287.1A.fwd and sAd4287.1A.rev, SEQ ID NOs: 52 and53, respectively) were designed to obtain the first 460 nucleotides ofsAd4287 by PCR, with PacI and SaII at the 5′- and 3′-end of theresulting PCR product, respectively. A second set of primers(sAd4287.1B.fwd and sAd4287.1B.rev, SEQ ID NOs: 54 and 55, respectively)was designed to obtain pIX (nt 2966) through 2.5 kb upstream (nt 5466),with AfIII and PacI designed on the 5′- and 3′-end, respectively. Athird set of PCR primers (sAd4287.TGC.fwd and sAd4287.TGC.rev, SEQ IDNOs: 56 and 57, respectively) were designed to obtain the transgenecassette from AdApter plasmid pAdApt26.Empty (Abbink, et al. J. Virol.81(9): 4654-4663, 2007) from start of the CMV to end of the polyA with aSaII and AfIII site designed on the 5′- and 3′-end, respectively. Thesethree PCR fragments were ligated together with the pAdApt bacterialbackbone obtained by PacI digestion from pAdApt26 in a 4-point ligation,resulting in sAdApt4287.Empty (SEQ ID NO: 34). A schematic map ofsAdApt4287.Empty is depicted in FIG. 2. This adapter plasmid containsleft-end sAd4287 sequences (1-460 and 2966-5466) with the E1 regionreplaced by an expression/transgene cassette including the CMV promoter.

Generation of pBr/sAd4287.pIX-pV

To enable cloning of an sAd4287 HpaI-HindIII restriction fragment, whichencompasses the 52K protein of sAd4287, a new plasmid was generated byinserting two PCR fragments in a pBr backbone. For this, primers (SEQ IDNOs: 58 and 59) were designed to obtain a PCR fragment from start of pIXover the HpaI site in wild-type sAd4287 (nt 2966 to nt 8311) with a PacIand a SbfI designed on the 5′- and 3′-end, respectively. A second PCRfragment was generated from HindIII (nt 12761) to the end of PCR (nt16679), with a SbfI and PacI site designed on the 5′- and 3′-end,respectively. The second PCR fragment was generated using a secondprimer set (SEQ ID NOs: 60 and 61). These PCR fragments were ligated(PacI-SbfI-PacI) into a pBr backbone, obtained from pBr/Ad26.SfiI (see,e.g., WO 2007/104792) by PacI digestion, resulting in thepBr/sAd4287.pIX-pV shuttle vector. Finally, the sAd4287 HpaI-HindIIIrestriction fragment obtained from the sAd4287 wild-type genome wasligated into the pBr/sAd4287.pIX-pV shuttle vector digested withHpaI-HindIII, resulting in the complete pBr/sAd4287.pIX-pV plasmid (SEQID NO: 35). A schematic map of pBr/sAd4287.pIX-pV is depicted in FIG. 3.

Generation of pBr/sAd4287.PsiI-rITR

pBr/sAd4287.PsiI-rITR contains sAd4287 sequences from the PsiI site atnucleotide 14053 to the end of the right inverted terminal repeat(rITR). To enable cloning of this sequence first a new plasmid wasgenerated by inserting two PCR fragments in a pBr backbone. The two PCRfragments were generated such that they could be ligated together andcloned into a pBr-based backbone using the PacI restriction site.Primers were designed to obtain a PCR fragment from before PsiI site atnt 14053 to ˜4 kb upstream over the NdeI site (nt 18186) at nt 18234,with a PacI and a SbfI site designed on the 5′- and 3′-end,respectively. A second set of primers was designed to obtain a PCRfragment from before PmeI site at nt30022 until the end of rITR atnt35079, with an SbfI and PacI site designed at the 5′- and 3′-end,respectively. The sequences of the primers used to generate these twoPCR fragments is set forth in SEQ ID NOs: 62-65. These PCR fragmentswere ligated into a pBr backbone obtained from pBr/Ad26.SfiI byPacI-SbfI digestion, resulting in the pBr/sAd4287.PsiI-rITR shuttlevector. Finally, the NotI-AsiSI fragment (nt 16639-nt 34032) wasobtained from the wild-type sAd4287 genome and ligated into thepBr/sAd4287.PsiI.rITR shuttle vector, resulting in the completepBr/sAd4287.PsiI-rITR plasmid (SEQ ID NO: 36). A schematic map ofpBr/sAd4287.PsiI-rITR is depicted in FIG. 4.

Generation of pBr/sAd4287.PsiI-rITR.dE3

pBr/sAd4287.PsiI-rITR was modified to delete part of the E3 region,which spans approximately nt 25973 to nt 28596 of sAd4287, and which isnot required for replication and packaging of the adenoviral particle.To create the pBr/sAd4287.PsiI-rITR.dE3, two PCR fragments weregenerated. The first PCR fragment contained the pVIII from AscI to 140bp after the polyA of pVIII (nt 8291-11192). The forward primer (SEQ IDNO: 66) was directed against the ApaLI in 100K and the reverse primer(SEQ ID NO: 67) has a SpeI site designed in it. The second PCR containsthe Fiber region starting 100 bp before the polyA of the E3 region untilthe unique XbaI restriction site in the Fiber-2 region (nt 13177-14824).The forward primer, directed 100 bp in front of the polyA of E3, willhave a SpeI site designed in it (SEQ ID NO: 68). The reverse primer wasdirected to the XbaI site (SEQ ID NO: 69). These two PCR fragments wereligated into pBr/sAd4287.PsiI-rITR with a 3-point ligation, withAscI-SpeI-XbaI, to generate pBr/sAd4287.PsiI-rITR.dE3 (SEQ ID NO: 37).FIG. 5 depicts a schematic map of pBr/sAd4287.PsiI-rITR.dE3 as well asan overview of the cloning strategy set forth above to generate theE3-deleted plasmid.

Generation of pBr/sAd4287.PsiI-rITR.dE3.dE4

pBr/sAd4287.PsiI-rITR.dE3 was modified to delete part of the E4 region,which spans approximately nt 31852 to nt 34752 of sAd4287, andspecifically E4orf1-E4orf4. The modified plasmid,pBr/sAd4287.PsiI-rITR.dE3.dE4 (SEQ ID NO: 38), resulted in an enlargedcloning capacity with a 1409 bp gain of space. To create thepBr/sAd4287.PsiI-rITR.dE3.dE4, two PCR products were generated. Thefirst PCR fragment starts at the XbaI site until the start of E4orf6.The sequences of the forward and reverse primers used to generate thisfirst PCR fragment are set forth in SEQ ID NOs: 72 and 73, respectively.The second PCR fragment starts directly in front of the E4orf1 until theNotI site. The sequences of the forward and reverse primers used tooperate this second PCR fragment are set forth in SEQ ID NOs: 74 and 75,respectively. These PCR fragments have 30-bp overlaps with flankingregions at the XbaI and Not I site and a 15-bp overlap with each other(30 bp total). The PCR fragments were assembled intopBr/sAd4287.PsiI-rITR.dE3 digested with XbaI and NotI by Gibson Assembly(New England BioLabs), resulting in pBr/sAd4287.PsiI-rITR.dE3.dE4. FIG.6 depicts a schematic map of pBr/sAd4287.PsiI-rITR.dE3.dE4 relative topBr/sAd4287.PsiI-rITR.dE3.

Generation of sAdApt4287.E1btg.Empty

To clone the E1 region of sAd4287 (approximately nt 474 to nt 3085 ofSEQ ID NO: 1) into sAdApt4257.Empty for the purposes of producingreplication-competent sAd4287 (rcsAd4287), a PCR fragment was generatedfrom the wild-type sAd4287 with the forward primer (SEQ ID NO: 70)starting ˜30 bp before the NgoMIV site in the lITR region until ˜10 bpafter the polyA of the E1 region (nt 218 to nt 3137). The reverse primer(SEQ ID NO: 71) has a ˜30 bp overlap with the start of the CMV promoterin the sAdApt4287.Empty and includes the SaII restriction site. This PCRfragment was cloned into sAdApt4257.Empty, digested with NgoMIV andSaII, with Gibson Assembly (New England BioLabs), resulting insAdApt4287.E1btg.Empty (SEQ ID NO: 39). A schematic map ofsAdApt4287.E1btg.Empty and the cloning strategy described above isdepicted in FIG. 7.

Example 3. Sequence of Simian Adenovirus sAd4310 #13-1 (sAd4310A)

The total genome sequence of simian adenovirus sAd4310 #13-1 (sAd4310A)was determined as described above for sAd4287. The obtained sequence ofthe sAd4310A genome (34391 nucleotides) is given as SEQ ID NO: 2. Aschematic map of the genome structure of sAd4310A is depicted in FIG. 8.Using the full genomic sequence in an NCBI web-based BLAST search, themost closely related virus to sAd4310A was identified as simianadenovirus 1 (sAd1) ATCC VR-195 (query coverage: 97%; maximum identity:98%). NCBI web-based BLAST searches were also performed to assesshomology of three major capsid proteins of sAd4310A (fiber-1, fiber-2,and hexon proteins). The most closely related protein to sAd4310Afiber-1 was identified as sAd1 fiber-1 (query coverage: 100%; maximumidentity: 99%). The most closely related protein to sAd4310A fiber-2 wasidentified as sAd1 fiber-2 (query coverage: 100%; maximum identity:99%). The most closely related protein to sAd4310A hexon was identifiedas human Ad31 hexon (query coverage: 100%; maximum identity: 87%).

Example 4. Generation of Recombinant sAd4310A Viruses

Here, the construction of an sAd4310A plasmid-based system to generaterecombinant sAd4310A vectors in a safe and efficient manner isdescribed. The plasmid system consists of a first plasmid, referred toas an adapter plasmid, which contains sAd4310A nucleotides 1 to 461including the left inverted terminal repeat (lITR) and packaging signal,an expression cassette and an sAd4310A fragment corresponding tonucleotides 2903 to 5410. The expression cassette comprises the humanCMV promoter, a multiple cloning site (MCS), and the SV40polyadenylation signal (polyA) as previously described (see, e.g., WO00/70071). The adapter plasmid is based on pAdApt26.Empty (Abbink, etal. J. Virol. 81(9): 4654-4663, 2007), albeit now generated to comprisethe sAd4310A-derived sequences instead of the Ad26-derived sequences.Furthermore, the system consists of other plasmids together constitutingsAd4310A sequences between nucleotide 2903 and 34391 that may be deletedfor E1 region (nt 474 to nt 3088 of SEQ ID NO: 2), E3 region (nt 25915to nt 28496 of SEC) ID NO: 2), and/or E4 region (nt 31750 to nt 34048 ofSEQ ID NO: 2) sequences.

Generation of Adapter Plasmid sAdApt4310A.Empty

Plasmids that were used for harboring the sAd4310A sequences wereprepared. Primers (sAd4310A.1A.fwd and sAd4310A.1A.rev, SEQ ID NOs: 76and 77, respectively) were designed to obtain the first 461 nucleotidesof sAd4310A by PCR, with PacI and SaII at the 5′- and 3′-end of theresulting PCR product, respectively. A second set of primers(sAd4310A.1B.fwd and sAd4310A.1B.rev, SEQ ID NOs: 78 and 79,respectively) was designed to obtain pIX (nt 2903) through approximately2.5 kb upstream (nt 5410), with AfIII and PacI designed on the 5′- and3′-end, respectively. A third set of PCR primers (sAd4310A.TGC.fwd andsAd4310A.TGC.rev, SEQ ID NOs: 80 and 81, respectively) were designed toobtain the transgene cassette from AdApter plasmid pAdApt26.Empty(Abbink, et al. J. Virol. 81(9): 4654-4663, 2007) from start of the CMVto end of the polyA with a SaII and AfIII site designed on the 5′- and3′-end, respectively. These three PCR fragments were ligated togetherwith the pAdApt bacterial backbone obtained by PacI digestion frompAdApt26 in a 4-point ligation, resulting in sAdApt4310A.Empty (SEQ IDNO: 40). A schematic map of sAdApt4310A.Empty is depicted in FIG. 9.This adapter plasmid contains left-end sAd4310A sequences (1-451 and2903-5410) with the E1 region replaced by an expression/transgenecassette including the CMV promoter.

Generation of pBr/sAd4310A.pIX-pV

To enable cloning of an sAd4310A SrfI-SnaBI restriction fragment, whichencompasses the 52K protein of sAd4310A, a new plasmid was generated byinserting two PCR fragments in a pBr backbone. For this, primers (SEQ IDNOs: 82 and 83) were designed to obtain a PCR fragment from start of pIXover the SrfI site in wild-type sAd4310A (nt 2903 to nt 7224) with aPacI and a SbfI designed on the 5′- and 3′-end, respectively. A secondPCR fragment was generated tram SnaBI (nt 12098) in pIIIa to pVI (nt17365), with a SbfI and PacI site designed on the 5′- and 3′-end,respectively. The second PCR fragment was generated using a secondprimer set (SEQ ID NOs: 84 and 85). These PCR fragments were ligated(PacI-SbfI-PacI) into a pBr backbone, obtained from pBr/Ad26.SfiI (see,e.g., WO 2007/104792) by PacI digestion, resulting in thepBr/sAd4310A.pIX-pV shuttle vector. Finally, the sAd4310A SrfI-SnaBIrestriction fragment obtained from the sAd4310A wild-type genome wasligated into the pBr/sAd4310A.pIX-pV shuttle vector digested withSrfI-SnaBI, resulting in the complete pBr/sAd4310A.pIX-pV plasmid (SEQID NO: 41). A schematic map of pBr/sAd4310A.pIX-pV is depicted in FIG.10.

Generation of pBr/sAd4310A.RSrII-rITR

pBr/sAd431 0A.RsrII-rITR contains sAd431 0A sequences from the RsrIIsite at nucleotide 14882 to the end of the right inverted terminalrepeat (rITR) at nucleotide 34391. To enable cloning of this sequencefirst a new plasmid was generated by inserting two PCR fragments in apBr backbone. The two PCR fragments were generated such that they couldbe ligated together and cloned into a pBr-based backbone using the Pacirestriction site. Primers (sAd431 0A.3A.fwd and sAd431 0A.3A.rev, SEQ IDNOs: 86 and 87, respectively) were designed to obtain a PCR fragmentfrom the RsrII site at nt 14882 to 4.5 kb upstream over the Sal I site(nt 19189) to nt 19224, with a Paci and a SbfI site designed on the 5′and 3′-end, respectively. A second set of primers (sAd431 0A.3B.fwd andsAd431 0A.3B.rev, SEQ ID NOs: 88 and 89, respectively) was designed toobtain a PCR fragment from before the PmeI site at nt 29829 until theend of the rITR at nt 34391, with an SbfI and Paci site designed at the5′- and 3′-end, respectively. These PCR fragments were ligated into aTOPO® vector using the commercially available ZERO BLUNT® TOPO® PCRCloning Kit (Invitrogen). The two PCR fragments were digested as PCRfragments or from the TOPO® clone with Paci and SbfI and subsequentlyligated into a pBr backbone obtained from pBr/Ad26.SfiI digested withPaci. Finally, SalI-XbaI fragment (nt 19190-nt 30014) was obtained fromthe wild-type sAd431 0A genome and ligated into the pBr/sAd4310A.RsrII.rITR shuttle vector, resulting in the complete pBr/sAd4310A.RsrII-rITR plasmid (SEQ ID NO: 42). A schematic map of pBr/sAd4310A.RsrII-rITR is depicted in FIG. 11.

Generation of pBr/sAd4310A.RsrII-rITR.dE3

pBr/sAd4310A.RsrII-rITR was modified to delete part of the E3 region,which spans approximately nt 25915 to nt 28496 of sAd4310A, and which isnot required for replication and packaging of the adenoviral particle.To create the pBr/sAd4310A.RsrII-rITR.dE3 with Gibson Assembly, two PCRfragments were generated. The first PCR fragment (dE3AG) contained fromapproximately 50 bp before the SfiI site at nt 7644 to 140 bp after thepolyA of pVIII. The forward primer and reverse primer have sequences setforth in SEQ ID NOs: 90 and 91, respectively, wherein the reverse primerwas designed to have an approximately 25-bp overlap with the second PCRfragment. The second PCR fragment (dE3BG) starts at nt 14641(approximately 100 bp before the polyA of the E3 region) untilapproximately 50 bp after the XbaI site at nt 16252. The forward primerand reverse primer for the second PCR have sequences set forth in SEQ IDNOs: 92 and 93, respectively, wherein the forward primer was designed tohave an approximately 25-bp overlap with the first PCR fragment. The twoPCR fragments were assembled with Gibson Assembly, with thepBr/sAd4310A.RsrII.rITR digested with SfiI and XbaI. The resultingplasmid, pBr/sAd4310A.RsrII.rITR.dE3 (SEQ ID NO: 43), is depicted inFIG. 12, along with the parental plasmid, pDr/sAd4310A.RsrII.rITR.

Generation of pBr/sAd4310A.RsrII-rITR.dE3.dE4

pBr/sAd4310A.RsrII-rITR.dE3 was modified to delete part of the E4region, which spans approximately nt 31750 to nt 34048 of sAd4310A, andspecifically E4orf1-E4orf4. The modified plasmid,pBr/sAd4310A.RsrII-rITR.CE3.dE4 (SEQ ID NO: 44), resulted in an enlargedcloning capacity with a 1394 bp pain of space. To create thepBr/sAd4310A.RsrII-rITR.dE3.dE4 plasmid, two PCR products weregenerated. The first PCR fragment starts at the XbaI site until thestart of E4orf6. The sequences of the forward and reverse primers usedto generate this first PCR fragment are set forth in SEQ ID NOs: 96 and97, respectively. The second PCR fragment starts directly in front ofthe E4orf1 until the NotI site. The sequences of the forward and reverseprimers used to generate this second PCR fragment are set forth in SEQID NOs: 98 and 99, respectively. These PCR fragments have 30-bp overlapswith flanking regions at the XbaI and Not I site and a 15-bp overlapwith each other (30 bp total). The PCR fragments were assembled byGibson Assembly (New England BioLabs) into pBr/sAd4310A.RsrII-rITR.dE3digested with XbaI and NotI, resulting inpBr/sAd4310A.RsrII-rITR.dE3.dE4 (SEQ ID NO: 44). FIG. 13 depicts aschematic map of pBr/sAd4310A.RsrII-rITR.dE3.dE4 relative to theparental plasmid pBr/sAd4310A.RsrII-rITR.dE3.

Generation of sAdApt4310A.E1btg.Empty

To clone the E1 region of sAd4310A (nt 474 to nt 3088 of SEQ ID NO: 2)into sAdApt4310A.Empty for the purposes of producingreplication-competent sAd4310A (rcsAd4310A), a PCR fragment wasgenerated from the wild-type sAd4310A with the forward primer (SEQ IDNO: 94) starting ˜40 bp before the BstZ17I site in the lITR region ˜10bp after the polyA of the E1 region (nt 150 to nt 3131). The reverseprimer (SEQ ID NO: 95) has a ˜30 bp overlap with the start of the CMVpromoter in the sAdApt4310A.Empty and includes the SaII restrictionsite. This PCR fragment was cloned into sAdApt4310A.Empty, digested withBstZ17I and SaII, with Gibson Assembly (New England BioLabs), resultingin sAdApt4310A.E1btg.Empty (SEQ ID NO: 45). A schematic map ofsAdApt4310A.E1btg.Empty and the cloning strategy described above isdepicted in FIG. 14.

Example 5. Sequence of Simian Adenovirus sAd4312

The total genome sequence of simian adenovirus sAd4312 was determined asdescribed above for sAd4287 and sAd4310A. The obtained sequence of thesAd4312 genome (34475 nucleotides) is given as SEQ ID NO: 3. A schematicmap of the genome structure of sAd4312 is depicted in FIG. 15. Using thefull genomic sequence in an NCBI web-based BLAST search, the mostclosely related virus to sAd4312 was identified as simian adenovirus 1(sAd1) ATCC VR-195 (query coverage: 90%; maximum identity: 98%). NCBIweb-based BLAST searches were also performed to assess homology of threemajor capsid proteins of sAd4312 (fiber-1, fiber-2, and hexon proteins).The most closely related protein to sAd4312 fiber-1 was identified ashuman Ad52 fiber-1 (query coverage: 100%; maximum identity: 99%). Themost closely related protein to sAd4312 fiber-2 was identified as sAd7lone fiber (query coverage: 99%; maximum identity: 73%). The mostclosely related protein to sAd4312 hexon was identified as human Ad40hexon (query coverage: 100%; maximum identity: 89%).

Example 6. Generation of Recombinant sAd4312 Viruses

Here, the construction of an sAd4312 plasmid-based system to generaterecombinant sAd4312 vectors in a safe and efficient manner is described.The plasmid system consists of a first plasmid, referred to as anadapter plasmid, which contains sAd4312 nucleotides 1 to 472 includingthe left inverted terminal repeat (lITR) and packaging signal, anexpression cassette and an sAd4312 fragment corresponding to nucleotides2939 to 5510. The expression cassette comprises the human CMV promoter,a multiple cloning site (MCS), and the SV40 polyadenylation signal(polyA) as previously described (see, e.g., WO 00/70071). The adapterplasmid is based on pAdApt26.Empty (Abbink, et al. J. Virol. 81(9):4654-4663, 2007), albeit now generated to comprise the sAd4312-derivedsequences instead of the Ad26-derived sequences. Furthermore, the systemconsists of other plasmids together constituting sAd4312 sequencesbetween nucleotide 2939 and 344475 that may be deleted for E1 region (nt487 to nt 3100 of SEQ ID NO: 3), E3 region (nt 25947 to nt 28561 SEQ IDNO: 3), and/or E4 region (nt 31818 to nt 34116 SEQ ID NO: 3) sequences.

Generation of Adapter Plasmid sAdApt4312.Empty

Plasmids that were used for harboring the sAd4312 sequences wereprepared. Primers (sAd4312.1A.fwd and sAd4312.1A.rev, SEQ ID NOs: 100and 101, respectively) were designed to obtain the first 472 nucleotidesof sAd4312 by PCR, with PacI and SaII at the 5′- and 3′-end of theresulting PCR product, respectively. A second set of primers(sAd4312.1B.fwd and sAd4312.1B.rev, SEQ ID NOs: 102 and 103,respectively) was designed to obtain pIX (nt 2939) through approximately2.5 kb upstream (nt 5510), with AfIII and PacI designed on the 5- and3′-end, respectively. A third set of PCR primers (sAd4312.TGC.fwd andsAd4312.TGC.rev, SEQ ID NOs: 104 and 105, respectively) were designed toobtain the transgene cassette from AdApter plasmid pAdApt26.Empty(Abbink, et al. J. Virol. 81(9): 4654-4663, 2007) from start of the PCRto end of the polyA with a SaII and AfIII site designed on the 5- and3′-end, respectively. These three PCR fragments were ligated togetherwith the pAdApt bacterial backbone obtained by PacI digestion frompAdApt26 in a 4-point ligation, resulting in sAdApt4312.Empty (SEQ IDNO: 46). A schematic map of sAdApt4312.Empty is depicted in FIG. 16.This adapter plasmid contains left-end sAd4312 sequences (1-472 and2939-5510) with the E1 region replaced by an expression/transgenecassette including the CMV promoter.

Generation of pBr/sAd4312.pIX-pV

To enable cloning of an sAd4312 BsiWI-BsiWI restriction fragment, a newplasmid was generated by inserting two PCR fragments in a pBr backbone.For this, primers (SEQ ID NOs: 106 and 107) were designed to obtain aPCR fragment from start of pIX over the BsiWI site in wild-type sAd4312(nt 2939 to nt 6791) with a Paci and a NdeI designed on the 5′- and3′-end, respectively. A second PCR fragment was generated from pV (nt15564) to the RsrII site at the end of pVI (nt 17698), with a NdeI andPaci site designed on the 5′- and 3′-end, respectively. The second PCRfragment was generated using a second primer set (SEQ ID NOs: 108 and109). These PCR fragments were cloned into a TOPO® vector using thecommercially available ZERO BLUNT® TOPO® PCR Cloning Kit (Invitrogen),resulting in the pBr/sAd4312.pIX-pV shuttle vector. Finally, the sAd4312BsiWI-BsiWI restriction fragment obtained from the sAd4312 wild-typegenome was ligated into the pBr/sAd4312.pIX-pV shuttle vector digestedwith BsiWI and screened for orientation, resulting in the completepBr/sAd4312.pIX-pV plasmid (SEQ ID NO: 47). A schematic map ofpBr/sAd4312.pIX-pV is depicted in FIG. 17.

Generation of pBr/sAd4312.pV-rITR

pBr/sAd4312.pV-rITR contains sAd4312 sequences from the start of pV atnucleotide 15215 to the end of the right inverted terminal repeat (rITR)at nucleotide 34475. To enable cloning of this sequence first a newplasmid was generated by inserting two PCR fragments in a pBr backbone.The two PCR fragments were generated such that they could be ligatedtogether and cloned into a pBr-based backbone using the Paci restrictionsite. Primers (sAd4312.3A.fwd and sAd4312.3A.rev, SEQ ID NOs: 110 and111, respectively) were designed to obtain a PCR fragment from the startof pV at nt 15215 to 2.5 kb upstream over the RsrII site to nt 17698,with a Paci and a SbfI site designed on the 5′- and 3′end, respectively.A second set of primers (sAd4312.3B.fwd and sAd4312.3B.rev, SEQ ID NOs:112 and 113, respectively) was designed to obtain a PCR fragment frombefore the XbaI site at nt 31015 until the end of the rITR at nt 34475,with an SbfI and Paci site designed at the 5′- and 3′-end, respectively.These PCR fragments were ligated into a TOPO® vector using thecommercially available ZERO BLUNT® TOPO® PCR Cloning Kit (Invitrogen).The two PCR fragments were digested from the TOPO® clones with SbfI andPaci and subsequently ligated into a pBr backbone obtained frompBr/Ad26.SfiI digested with Paci, resulting in the pBr/sAd4312.pV-rITRshuttle vector. Finally, the NotI-XbaI fragment (nt 16412-nt 31083) wasobtained from the wild-type sAd4312 genome and ligated into thepBr/sAd4312.pV-rITR shuttle vector, resulting in the completepBr/sAd4312.pV-rITR plasmid (SEQ ID NO: 48). A schematic 1 o map ofpBr/sAd4312.pV-rITR is depicted in FIG. 18.

Generation of pBr/sAd4312.pV-rITR.dE3

pBr/sAd4312.pV-rITR was modified to delete part of the E3 region, whichspans approximately nt 487 to nt 3100 of sAd4312, and which is notrequired for replication and packaging of the adenoviral particle. Tocreate the pBr/sAd4312.pV-rITR.dE3, two PCR fragments were generated.The first PCR fragment contains the pVIII from AscI to 140 bp after thepolyA of pVIII (nt 9859 to nt 12302). The forward primer(sAd4312.dE3A.fwd, SEQ ID NO: 114) is directed against the AscI in 100K,and the reverse primer (sAd4312.dE3A.rev, SEQ ID NO: 115) has a SpeIsite designed in it.

The second PCR contains the fiber region starting 100 bp before thepolyA of the E3 region until the unique restriction site, XbaI, in thefiber-2 region (nt 14378 to nt 17020). The forward primer(sAd4312.dE3B.fwd, SEQ ID NO: 116), directed 100 bp in front of thepolyA of E3, has a SpeI site designed in it. The reverse primer(sAd4312.dE3B.fwd, SEQ ID NO: 117) is directed to the XbaI site. Thesetwo PCR fragments were ligated into pBr/sAd4312.pV-rITR with a 3-pointligation, with AscI-SpeI-XbaI. The resulting plasmid,pBr/sAd4312.pV-rITR.dE3 (SEQ ID NO: 49), is depicted in FIG. 19, alongwith the parental plasmid, pBr/sAd4312.pV-rITR.

Generation of pBr/sAd4312.pV-rITR.dE3.dE4

pBr/sAd4312/pV-rITR.dE3 was modified to delete part of the E4 region,which spans approximately nt 25947 to nt 28561 of sAd4312, andspecifically E4orf1-E4orf4. The modified plasmid,pBr/sAd4312.pV-rITR.dE3.dE4 (SEQ ID NO: 50), resulted in an enlargedcloning capacity with a 1393 bp gain of space. To create thepBr/sAd4312.pV-rITR.dE3.dE4 plasmid, two PCR products were generated.The first PCR fragment starts at the NdeI site until the start ofE4orf6. The sequences of the forward and reverse primers used togenerate this first PCR fragment are set forth in SEQ ID NOs: 120 and121, respectively. The second PCR fragment starts directly in front ofthe E4orf1 until the NotI site. The sequences of the forward and reverseprimers used to generate this second PCR fragment are set forth in SEQID NOs: 122 and 123, respectively. These PCR fragments have 30-bpoverlaps with flanking regions at the NdeI and NotI site and a 15-bpoverlap with each other (30 bp total). The PCR fragments were assembledinto pBr/sAd4312.pV-rITR.dE3 digested with XbaI and NotI, resulting inpBr/sAd4312.pV-rITR.dE3.dE4 (SEQ ID NO: 50). FIG. 20 depicts a schematicmap of pBr/sAd4312.pV-rITR.dE3.dE4 and that of the parental plasmid,pBr/sAd4312.pV-rITR.dE3.

Generation of sAdApt4312.E1btg.Empty

To clone the E1 region of sAd4312 (nt 487 to 3100 SEQ ID NO: 3) intosAdApt4312.Empty for the purposes of producing replication-competentsAd4312 (rcsAd4312), a PCR fragment was generated from the wild-typesAd4312 which included the complete E1 region of sAd4312. The forwardprimer (SEQ ID NO: 118) is directed to ˜40 bp in front of the firstBstZ17I site in the lITR region. The reverse primer (SEQ ID NO: 119) hasa ˜30 bp overlap with the start of the CMV promoter in thesAdApt4312.Empty. The generated PCR fragment was cloned intosAdApt4312.Empty, digested with BstZ17I and SaII, with Gibson Assembly(New England BioLabs), resulting in sAdApt4312.E1btg.Empty (SEQ ID NO:51). In this cloning step, only the AdApt plasmid was digested; the PCRproduct was not digested with restriction enzymes. A schematic map ofsAdApt4312.E1btg.Empty and the cloning strategy described above isdepicted in FIG. 21.

Example 7. Seroprevalence of sAd4287, sAd4310A, and sAd4312 insub-Saharan Humans and Rhesus Monkeys

We next evaluated sAd4287, sAd4310A, and sAd4312 titers in 144sub-Saharan humans and 108 rhesus monkeys (FIGS. 22A-22C).Adenovirus-specific neutralizing antibody (NAb) titers were determinedby luciferase-based virus neutralization assays as previously described(Sprangers et al. J. Clin. Microbiol. 41: 5046-5052, 2003; Barouch etal. Vaccine. 29: 5203-5209, 2011). Titers of <18 are regarded asnegative by this assay, 18-200 is low, 201-1000 is high, and >1000 isconsidered very high. It is suspected that titers >200 will likely besuppressive, according to data known in the art. Representative piecharts summarizing the relative number of individuals (humans ormonkeys) that fall within each of the four titer categories are depictedfor each of the three adenoviruses tested (see FIGS. 22A-22C).

The results of the seroprevalence studies clearly indicate that themajority of both sub-Saharan humans and rhesus monkeys tested exhibitednegative (<18) or low (18-200) NAb titers for each of the threeadenoviruses tested (sAd4287, sAd4310A, and sAd4312). Theseseroprevalence studies indicate that the sAd4287, sAd4310A, and sAd4312vectors have extremely and surprisingly low seroprevalence in humanpopulations (e.g., sub-Saharan human populations) and monkey populations(e.g., rhesus monkey populations). The extremely low seroprevalence ofthe sAd vectors of the invention are in marked contrast to therelatively high seroprevalence of Ad5 in human populations. Accordingly,these studies indicate a distinct advantage of using a vaccinecomprising all or a portion of a recombinant sAd4287, sAd4310A, andsAd4312, as the neutralizing activities in the majority of both humansand monkeys alike are unlikely to hamper the efficacy of the vaccine.

Example 8. Determination of Cellular Responses to RecombinantAdenoviruses of the Invention in Mice

We next studied whether recombinant replication-defective adenovirusesbased on simian adenoviruses of the invention (e.g., sAd4287 orsAd4310A) were able to elicit a significant immune response in vivo. Forthis, vectors were generated that all contained the SIVmac239 Gag insertfrom Simian Immunodeficiency Virus (SIV). Recombinant DNA, such as therequired adapter plasmids, and the recombinant viruses were generatedgenerally as described (Lemckert et al. J. Virol. 79:9694-9701, 2005).

C57BL/6 mice were injected intramuscularly with different amounts ofviral vectors: 10⁷, 10⁸, and 10⁹ viral particles (vp). All vaccinationprocedures and cellular immune responses were performed and measured byassessing the CD8⁺ T cell response via D^(b)/AL11 tetramer bindingassays as previously described (Barouch et al. J. Immunol.172:6290-6297, 2004). Tetrameric H-2Db complexes folded around theimmunodominant SIV Gag AL11 epitope (AAVKNWMTQTL; SEQ ID NO: 124) (Liuet al., J. Viral. 80: 11991-11997, 2006) were prepared and SIVGag-specific CD8⁺ T lymphocyte responses were measured on days 0, 7, 14,21, and 28 post-immunization. For immunogenicity experiments withsAd4287 and sAd4310A, the results are shown in FIGS. 23A and 23B. Fromthese results, it can be concluded that the adenoviral vectors of theinvention exhibit potent immunogenicity in mice, especially with 10⁸ or10⁹ vp doses.

To evaluate functional responses, splenocytes from day 28 were utilizedin IFN-γ ELISPOT assays. IFN-γ ELISPOT responses were measured tooverlapping Gag peptides (Gag), the dominant CD8+ T cell epitope AL11(AAVKNWMTQTL; SEQ ID NO: 124), the sub-dominant CD8⁺ T epitope KV9(KSLYNTVCV; SEQ ID NO: 125), and the CO4+ T cell epitope DD13(DRFYKSLRAEQTD; SEQ ID NO: 126) (Liu et al., J. Virol. 80: 11991-11997,2006) at 10⁷, 10⁸, and 10⁹ vp of viral vectors (sAd4287, sAd431 0A, andrcsAd4287). As depicted in FIGS. 24A-24C, the IFN-γ ELISPOT responsesincreased with increasing amounts of vp, and both Gag and AL11 responseswere elevated relative to the responses to KV9 or DD13 epitopes. Inaddition, these functional responses were elicited only whenreplication-defective adenoviruses of the invention were used (e.g.,sAd4287 and sAd4310A), but not when replication-competent adenovirusesof the invention were used (e.g., rcsAd4287). Collectively, the studiesof cellular responses to the recombinant adenoviral vectors of theinvention clearly indicate potent immunogenicity in mice.

The combination of low baseline anti-vector immunity (lowseroprevalence), potent immunogenicity, and novel biology suggests thatthe novel adenoviral vectors of the invention can be useful as novelvaccine candidates against human or veterinary pathogens, including, butnot limited to, HIV, SIV, cancer, malaria, and tuberculosis, in additionto utility in gene therapy and/or diagnostics.

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated as being incorporated by reference in theirentirety.

What is claimed is:
 1. A recombinant adenovirus comprising a nucleotidesequence having at least 90% sequence identity over the entire sequenceof any one of SEQ ID NOs: 10 and 12, or a complementary sequence to anucleotide sequence having at least 90% sequence identity over theentire sequence of any one of SEQ ID NOs: 10 and 12; wherein saidrecombinant adenovirus comprises a deletion in or of an E1 region, an E3region, and/or an E4 region, said deletion rendering said recombinantadenovirus a replication-defective virus.
 2. The recombinant adenovirusof claim 1, wherein said nucleotide sequence further comprises all or aportion of any one of SEQ ID NOs: 4, 6, 7, 9, 13, 15, 16, and 18, or acomplementary sequence to all or a portion of any one of SEQ ID NOs: 4,6, 7, 9, 13, 15, 16, and
 18. 3. The recombinant adenovirus of claim 1,further comprising a nucleotide sequence having at least 90% sequenceidentity to the sequence of any one of SEQ ID NOs: 1 and 3 or acomplementary sequence to a nucleotide sequence having at least 90%sequence identity over the entire sequence of any one of SEQ ID NOs: 1and
 3. 4. The recombinant adenovirus of claim 2, wherein said nucleotidesequence comprises the nucleotide sequence of any one of SEQ ID NOs:34-39 and 46-51.
 5. The recombinant adenovirus of claim 1, furthercomprising a heterologous nucleotide sequence encoding an antigenic ortherapeutic gene product of interest, or fragment thereof, wherein saidantigenic gene product, or fragment thereof, comprises a bacterial,viral, parasitic, or fungal protein, or fragment thereof.
 6. Therecombinant adenovirus of claim 5, wherein: (i) said bacterial protein,or fragment thereof, is from Mycobacterium tuberculosis, Mycobacteriumbovis, Mycobacterium africanum, Mycobacterium microti, Mycobacteriumleprae, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichiacoli, Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcusaureus, Francisella tularensis, Brucella, Burkholderia mallei, Yersiniapestis, Corynebacterium diphtheria, Neisseria meningitidis, Bordetellapertussis, Clostridium tetani, or Bacillus anthracis; (ii) saidparasitic protein, or fragment thereof, is from Toxoplasma gondii,Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae, Trypanosoma spp., or Legionella spp; or (iii) said fungalprotein, or fragment thereof, is from Aspergillus, Blastomycesdermatitidis, Candida, Coccidioides immitis, Cryptococcus neoformans,Histoplasma capsulatum var. capsulatum, Paracoccidioides brasiliensis,Sporothrix schenckii, Zygomycetes spp., Absidia corymbifera, Rhizomucorpusillus, or Rhizopus arrhizus.
 7. The recombinant adenovirus of claim5, wherein said viral protein, or fragment thereof, is from a viralfamily selected from the group consisting of Retroviridae, Flaviviridae,Arenaviridae, Bunyaviridae, Filoviridae, Togaviridae, Poxviridae,Herpesviridae, Orthomyxoviridae, Coronaviridae, Rhabdoviridae,Paramyxoviridae, Picornaviridae, Hepadnaviridae, Papillomaviridae,Parvoviridae, Astroviridae, Polyomaviridae, Calciviridae, andReoviridae, or said viral protein, or fragment thereof, is from humanimmunodeficiency virus (HIV), human papillomavirus (HPV), hepatitis Avirus (Hep A), hepatitis B virus (HBV), hepatitis C virus (HCV), Variolamajor, Variola minor, monkeypox virus, measles virus, rubella virus,mumps virus, varicella zoster virus (VZV), poliovirus, rabies virus,Japanese encephalitis virus, herpes simplex virus (HSV), cytomegalovirus(CMV), rotavirus, influenza, Ebola virus, yellow fever virus, Zikavirus, or Marburg virus.
 8. The recombinant adenovirus of claim 7,wherein said viral protein, or fragment thereof, from HIV is Gag, Pol,Env, Nef, Tat, Rev, Vif, Vpr, or Vpu.
 9. A method of treating a subjecthaving a disease, said method comprising administering the recombinantadenovirus of claim 5 to said subject.
 10. The method of claim 9,wherein said recombinant adenovirus comprises an antigenic gene product,or fragment thereof, that promotes an immune response in said subjectagainst an infective agent, wherein said infective agent is a bacterium,a virus, a parasite, or a fungus.
 11. The method of claim 9, wherein:(a) said subject is human; (b) said adenovirus is administeredintramuscularly; and/or (c) said adenovirus is administered as apharmaceutical composition comprising a pharmaceutically acceptablecarrier.
 12. The method of claim 11, wherein: (i) said subject isadministered at least one dose of said pharmaceutical composition; (ii)said subject is administered at least two doses of said pharmaceuticalcomposition; (iii) said pharmaceutical composition is administered tosaid subject as a prime boost.
 13. A method of producing a recombinantadenovirus comprising transfecting a cell with: (a) an isolatedpolynucleotide comprising a nucleotide sequence having at least 90%sequence identity over the entire sequence of any one of SEQ ID NOs: 10and 12, or a complementary sequence to a nucleotide sequence having atleast 90% sequence identity over the entire sequence of any one of SEQID NOs: 10 and 12 or (b) a recombinant vector comprising saidpolynucleotide; culturing said cell in a suitable medium to allowreplication of said polynucleotide or vector in said cell; andharvesting produced recombinant adenovirus from said medium and/or saidcell.
 14. The method of claim 13, wherein said cell is a bacterial,plant, or mammalian cell, wherein optionally said mammalian cell is aPER.55K cell or a Chinese hamster ovary (CHO) cell.
 15. The recombinantadenovirus of claim 5, wherein the viral gene product is an envelopeglycoprotein or fragment thereof.
 16. A method of inducing an immuneresponse against a flavivirus in a subject comprising administering therecombinant adenovirus of claim 5 to said subject, wherein the antigenicgene product, or fragment thereof, is a viral gene product from theflavivirus.
 17. The method of claim 16, wherein the viral gene productis an envelope glycoprotein or fragment thereof.
 18. The method of claim16, wherein the subject is a human.
 19. The method of claim 16, whereinthe adenovirus is administered intramuscularly.
 20. The method of claim16, wherein the adenovirus is administered as a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier.
 21. Themethod of claim 20, wherein the subject is administered at least one ortwo doses of the pharmaceutical composition, optionally wherein thepharmaceutical composition is administered to the subject as a primeboost.
 22. A method of inducing an immune response against a retrovirusin a subject comprising administering the recombinant adenovirus ofclaim 5 to the subject, wherein the antigenic gene product, or fragmentthereof, is a viral gene product from the retrovirus.
 23. The method ofclaim 22, wherein the retrovirus is human immunodeficiency virus (HIV).24. The method of claim 22, wherein the subject is a human.
 25. Themethod of claim 22, wherein the viral gene product is an envelopeglycoprotein or fragment thereof.
 26. The method of claim 25, whereinthe viral gene product is a protein or fragment thereof from HIV. 27.The method of claim 22, wherein the recombinant adenovirus isadministered intramuscularly.
 28. The method of claim 22, wherein therecombinant adenovirus is administered as a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier.
 29. The method ofclaim 28, wherein the subject is administered at least one or two dosesof the pharmaceutical composition, optionally wherein the pharmaceuticalcomposition is administered to the subject as a prime boost.